Methods of determining the health status of an individual

ABSTRACT

Methods of determining health status based on analysis of single cells in a sample or set of samples from an individual are described.

CROSS-REFERENCE

This application claims the benefit of the filing date of U.S. Ser. No.61,048,657 filed Apr. 29, 2008, this provisional application is herebyexpressly incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Even though there have been great gains in knowledge over the pastseveral decades in the fields of genetics and cellular and molecularbiology, this expansion of knowledge has not translated intocommensurate advances in the diagnosis or prognosis of disease, or theability to predict or assess response to therapy. New methods fordiagnosis and prognosis that harness the advances in the biologicsciences are needed.

SUMMARY OF THE INVENTION

One aspect of this invention provides a method for determining thestatus of an individual. In some embodiments, the invention providesmethods to determining the status of an individual by identifying a rarecell population associated with a status. In some embodiments, thestatus is a health status. In some embodiments, the invention provides amethod of predicting a change in a health status in an individual from afirst state to a second state comprising: determining the presence of afirst and second class of cells in a sample from the individual, thepresence being determined by a method comprising determining anactivation level of an intracellular activatable element in single cellsfrom said sample, classifying single cells into the first and secondclass, wherein at least one class is classified based on the activationlevel; calculating a ratio of the first and second class of cells andusing the ratio to predict said change in health status; and predictinga change in a health status in the individual from a first state to asecond state when said ratio exceeds a threshold number. In someembodiments, the threshold number expressed as a percentage is 30%. Insome embodiments, the threshold number expressed as a percentage is 5%.In some embodiments threshold number expressed as a percentage is 1%. Insome embodiments, the threshold number expressed as cell frequency is10⁻². In some embodiments, the threshold number expressed as cellfrequency is 10⁻³. In some embodiments, the threshold number expressedas cell frequency is 10⁻⁴.

In some embodiments, the second state is the location of an individualon a continuum that comprises normal, pre-pathological, and pathologicalstates. In some embodiments, the pathological state of the continuum isan immunologic, malignant, or proliferative disorder or a combinationthereof. In some embodiments, the status is a predicted response to atreatment for a pre-pathological or pathological condition, or aresponse to treatment for a pre-pathological or pathological condition.

In some embodiments, the pathological state is a malignant disorder. Insome embodiments, the malignant disorder is a solid tumor or ahematologic malignancy. In some embodiments, the malignant disorderincludes metastases. In some embodiments, the malignant disorder isnon-B cell lineage derived. In some embodiments, the non-B cell lineagederived malignant disorder is selected from the group consisting ofAcute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), non-B cellAcute Lymphocytic Leukemia (ALL), non-B cell lymphomas, myelodysplasticdisorders, myeloproliferative disorders, myelofibroses, polycythemias,thrombocythemias, and non-B atypical immune lymphoproliferations. Insome embodiments, the non-B cell lineage derived malignant disorder isAML.

In some embodiments, the pathological state is a malignant disorder thatis derived from a B cell or B cell lineage. In some embodiments, themalignant disorder is a B-Cell or B cell lineage derived disorder isselected from the group consisting of Chronic Lymphocytic Leukemia(CLL), B cell lymphocyte lineage leukemia, B cell lymphocyte lineagelymphoma, Multiple Myeloma, and plasma cell disorders. In someembodiments, the B-Cell or B cell lineage derived disorder is CLL.

In some embodiments, the methods of the invention further comprisepredicting a response to a treatment for a pre-pathological orpathological condition, or a response to treatment for apre-pathological or pathological condition.

In some embodiments, the activation levels of a plurality ofintracellular activatable elements in single cells are determined. Insome embodiments, the activation level of at least about 2, 3, 4, 5, 6,7, 8, 9, 10, or more than 10 intracellular (counting by whole numbers)activatable elements is determined.

In some embodiments, the plurality of cells obtained from the individualis first exposed to a modulator before determining said activationlevels of said activatable element(s). In some embodiments, theplurality of cells is divided into separate groups and each group issubjected to a different modulator.

In some embodiments, the sample from the individual is a blood sample.In some embodiments, the sample is a biopsy sample or a surgical sample.

In some embodiments, calculating a ratio of the classes of cellscomprises a determination of the number of cells in one or moreparticular classes of cells. In some embodiments, the status of theindividual is determined by a process comprising determining whether ornot the number of cells in one or more of said classes is greater than,less than, or equal to a threshold number. In some embodiments, thethreshold number of cells in one or more classes is about 0, 1, 5, 10,50, 100, 500, 1000, 10,000, 100,000, or 1,000,000. In some embodiments,determining the status of an individual comprises determining whether ornot the number of cells in a class is greater than a threshold number of0. In some embodiments, the class is a predefined class.

In some embodiments, the class is a class of cells wherein one or moreactivation levels of the cells are different when compared todeterminations made in healthy control samples, or when compared toprevious determinations made in a series of samples from saidindividual. In some embodiments, the one or more different activationlevels comprise one or more additional activation levels compared tohealthy controls or previous samples from said individual. In someembodiments, one or more different activation levels comprises one orfewer activation levels compared to healthy controls or previous samplesfrom said individual.

In some embodiments, the ratio is determined by comparing the number ofcells in one or more particular class or classes of cells to the numberof cells in one or more other class or classes of cells, or to the totalnumber of cells in the sample or a fraction of the sample. In someembodiments, the status is determined by a process comprisingdetermining whether or not said ratio is greater than, less than, orequal to a threshold number. In some embodiments, the threshold ratio,expressed as a percentage, is about 0%, 0.0000001%, 0.000001%, 0.00001%,0.0001%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, 5.0%, 10%, 20%,or 30%.

In some embodiments, the determination of a status in an individual isperformed on a plurality of samples from the individual. In someembodiments, the plurality of samples comprises samples from differentlocations in the individual, samples taken at different times from theindividual, samples treated in different ways prior to determining theactivation level, or a combination thereof. In some embodiments, theplurality of samples comprises a series of samples taken from theindividual at different times.

In some embodiments, the method further comprises determining of therate of change in the number of cells in one or more of said classes, ordetermining the rate of change of the ratio of the number of cells inone or more particular class or classes of cells to the number of cellsin one or more other class or classes of cells, or to the total numberof cells in the sample or a fraction of the sample. In some embodiments,the rate of change is expressed as the doubling time of said cells. Insome embodiments, the status is determined by a process comprisinganalyzing said rate of change.

In some embodiments, the method of determining the status of anindividual further comprises determining an appropriate course oftreatment for said individual based on said status of the individual. Insome embodiments, the appropriate course of treatment comprises watchfulwaiting, supportive therapy, initiating a therapy, not initiating atherapy, stopping, shortening, prolonging, or modifying an existingtherapy, adding an additional therapy to existing therapy, orcombinations of the foregoing. In some embodiments, therapy is selectedfrom the group consisting of surgical excision, transplantation, or theadministration of a physical, chemical, or biological agent, orcombinations thereof.

In some embodiments, one or more characteristics of the individual isdetermined, and the status of the individual is then determined based onboth quantitative analysis of classes of cells and the one or morecharacteristics of the individual. In some embodiments, thedetermination of an appropriate course of treatment is also based on oneor more characteristics of the individual. In some embodiments, the oneor more characteristics comprise physical characteristics, clinicalstatus, treatment characteristics, and biochemical/molecular markers.

In some embodiments, the modulator is an activator or an inhibitor. Insome embodiments, the modulator is a growth factor, cytokine, adhesionmolecule modulator, hormone, small molecule, polynucleotide, antibody,natural compound, lactone, chemotherapeutic agent, immune modulator,carbohydrate, protease, ion, reactive oxygen species, or radiation. Insome embodiments, the modulator is a B cell receptor modulator. In someembodiments, the B cell receptor modulator is a B cell receptoractivator. In some embodiments, the B cell receptor activator is across-linker of the B cell receptor complex or the B cell co-receptorcomplex.

In some embodiments, the cross-linker is an antibody or a molecularbinding entity. In some embodiments, the modulator is an inhibitor thatinhibits a cellular factor or a plurality of factors that participatesin a signaling cascade in the cell. In some embodiments, the inhibitoris a phosphatase inhibitor. In some embodiments, the phosphataseinhibitor is H₂O₂.

In some embodiments, the cells are further subjected to a secondmodulator. In some embodiments, the two modulators are a B cell receptoractivator and a phosphatase inhibitor. In some embodiments, themodulators are F(ab)₂IgM or biotinylated F(ab)₂IgM and H₂O₂.

In some embodiments, the activation state is selected from the groupconsisting of cleavage by extracellular or intracellular proteaseexposure, novel hetero-oligomer formation, glycosylation state,phosphorylation state, acetylation state, methylation state,biotinylation state, glutamylation state, glycylation state,hydroxylation state, isomerization state, prenylation state,myristoylation state, lipoylation state, phosphopantetheinylation state,sulfation state, ISGylation state, nitrosylation state, palmitoylationstate, SUMOylation state, ubiquitination state, neddylation state,citrullination state, deamidation state, disulfide bond formation state,proteolytic cleavage state, translocation state, changes in proteinturnover, multi-protein complex state, oxidation state, multi-lipidcomplex, and biochemical changes in cell membrane. In some embodiments,the activation state is a phosphorylation state. In some embodiments,the activatable element is selected from the group consisting ofproteins, carbohydrates, lipids, nucleic acids and metabolites. In someembodiments, the activatable element is a protein. In some embodiments,the protein is a protein subject to phosphorylation and/ordephosphorylation. In some embodiments, the protein is selected from thegroup consisting of kinases, phosphatases, lipid signaling molecules,adaptor/scaffold proteins, cytokines, cytokine regulators,ubiquitination enzymes, adhesion molecules, cytoskeletal proteins,heterotrimeric G proteins, small molecular weight GTPases, guaninenucleotide exchange factors, GTPase activating proteins, caspases,proteins involved in apoptosis, cell cycle regulators, molecularchaperones, metabolic enzymes, vesicular transport proteins,hydroxylases, isomerases, deacetylases, methylases, demethylases, tumorsuppressor genes, proteases, ion channels, molecular transporters,transcription factors/DNA binding factors, regulators of transcription,and regulators of translation. In some embodiments, the protein isselected from the group consisting of HER receptors, PDGF receptors, Kitreceptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors,Insulin receptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK,Jak1, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk,ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK,TGFβ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1, Wee1, Casein kinases,PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3, p90Rsks, p70S6Kinase, Prks,PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs,Chk1, Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3, IKKs, Cdks, Jnks, Erks,IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, Receptor proteintyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptortyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs),Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecularweight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases,Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C,PP1, PP5, inositol phosphatases, PTEN, SHIPs, myotubularins,phosphoinositide kinases, phospholipases, prostaglandin synthases,5-lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffoldproteins, Shc, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP),SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nck, Grb2 associated binder(GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-Cellleukemia family, IL-2, IL-4, IL-8, IL-6, interferon γ, interferon α,suppressors of cytokine signaling (SOCs), Cbl, SCF ubiquitination ligasecomplex, APC/C, adhesion molecules, integrins, Immunoglobulin-likeadhesion molecules, selectins, cadherins, catenins, focal adhesionkinase, p130CAS, fodrin, actin, paxillin, myosin, myosin bindingproteins, tubulin, eg5/KSP, CENPs, β-adrenergic receptors, muscarinicreceptors, adenylyl cyclase receptors, small molecular weight GTPases,H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam,Sos, Dbl, PRK, TSC1,2, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2,Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Bcl-2, Mcl-1,Bcl-XL, Bcl-w, Bcl-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk,Noxa, Puma, IAPs, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D,Cyclin E, Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecularchaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAaCarboxylase, ATP citrate lyase, nitric oxide synthase, caveolins,endosomal sorting complex required for transport (ESCRT) proteins,vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylasesPHD-1, 2 and 3, asparagine hydroxylase FIH transferases, Pin1 prolylisomerase, topoisomerases, deacetylases, Histone deacetylases, sirtuins,histone acetylases, CBP/P300 family, MYST family, ATF2, DNA methyltransferases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-1,p53, Hdm, PTEN, ubiquitin proteases, urokinase-type plasminogenactivator (uPA) and uPA receptor (uPAR) system, cathepsins,metalloproteinases, esterases, hydrolases, separase, potassium channels,sodium channels, multi-drug resistance proteins, P-Gycoprotein,nucleoside transporters, Ets, Elk, SMADs, Rel-A (p65-NFKB), CREB, NFAT,ATF-2, AFT, Myc, Fos, Sp1, Egr-1, T-bet, β-catenin, HIFs, FOXOs, E2Fs,SRFs, TCFs, Egr-1, β-catenin, FOXO STAT1, STAT 3, STAT 4, STAT 5, STAT6, p53, WT-1, HMGA, pS6, 4EPB-1, eIF4E-binding protein, RNA polymerase,initiation factors, and elongation factors. In some embodiments, theprotein is selected from the group consisting of Erk, Syk, Zap70, Lyn,Btk, BLNK, Cbl, PLCγ2, Akt, RelA, p38, S6. In some embodiments, theprotein is S6. In some embodiments, the activatable element isresponsive to a change in metabolic state, temperature, local ionconcentration, or heterologous protein expression.

In some embodiments, the activation level is determined by a processcomprising the binding of a binding element which is specific to aparticular activation state of the particular activatable element. Insome embodiments, the binding element comprises a protein. In someembodiments, the protein is an antibody. In some embodiments, theantibody binds to an activatable element selected from the groupconsisting of kinases, phosphatases, adaptor/scaffold proteins,ubiquitination enzymes, adhesion molecules, contractile proteins,cytoskeletal proteins, heterotrimeric G proteins, small molecular weightGTPases, guanine nucleotide exchange factors, GTPase activatingproteins, caspases and proteins involved in apoptosis, ion channels,molecular transporters, molecular chaperones, metabolic enzymes,vesicular transport proteins, hydroxylases, isomerases, transferases,deacetylases, methylases, demethylases, proteases, esterases,hydrolases, DNA binding proteins and transcription factors.

In some embodiments, the antibody binds to an activatable elementselected from the group consisting of HER receptors, PDGF receptors, Kitreceptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors,Insulin receptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK,Jak1, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk,ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK,TGFβ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1, Wee1, Casein kinases,PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3, p90Rsks, p70S6Kinase, Prks,PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs,Chk1, Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3, IKKs, Cdks, Jnks, Erks,IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, Receptor proteintyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptortyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs),Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecularweight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases,Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C,PP1, PP5, inositol phosphatases, PTEN, SHIPs, myotubularins,phosphoinositide kinases, phospholipases, prostaglandin synthases,5-lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffoldproteins, Shc, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP),SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nck, Grb2 associated binder(GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-Cellleukemia family, IL-2, IL-4, IL-8, IL-6, interferon γ, interferon α,suppressors of cytokine signaling (SOCs), Cbl, SCF ubiquitination ligasecomplex, APC/C, adhesion molecules, integrins, Immunoglobulin-likeadhesion molecules, selectins, cadherins, catenins, focal adhesionkinase, p130CAS, fodrin, actin, paxillin, myosin, myosin bindingproteins, tubulin, eg5/KSP, CENPs, β-adrenergic receptors, muscarinicreceptors, adenylyl cyclase receptors, small molecular weight GTPases,H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam,Sos, Dbl, PRK, TSC1,2, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2,Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Bcl-2, Mcl-1,Bcl-XL, Bcl-w, Bcl-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk,Noxa, Puma, IAPs, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D,Cyclin E, Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecularchaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAaCarboxylase, ATP citrate lyase, nitric oxide synthase, caveolins,endosomal sorting complex required for transport (ESCRT) proteins,vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylasesPHD-1, 2 and 3, asparagine hydroxylase FIH transferases, Pin1 prolylisomerase, topoisomerases, deacetylases, Histone deacetylases, sirtuins,histone acetylases, CBP/P300 family, MYST family, ATF2, DNA methyltransferases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-1,p53, Hdm, PTEN, ubiquitin proteases, urokinase-type plasminogenactivator (uPA) and uPA receptor (uPAR) system, cathepsins,metalloproteinases, esterases, hydrolases, separase, potassium channels,sodium channels, multi-drug resistance proteins, P-Gycoprotein,nucleoside transporters, Ets, Elk, SMADs, Rel-A (p65-NFKB), CREB, NFAT,ATF-2, AFT, Myc, Fos, Sp1, Egr-1, T-bet, β-catenin, HIFs, FOXOs, E2Fs,SRFs, TCFs, Egr-1, β-catenin, FOXO STAT1, STAT 3, STAT 4, STAT 5, STAT6, p53, WT-1, HMGA, pS6, 4EPB-1, eIF4E-binding protein, RNA polymerase,initiation factors, and elongation factors.

In some embodiments, the step of finding the activation level comprisesthe use of flow cytometry, immunofluorescence, confocal microscopy,immunohistochemistry, immunoelectronmicroscopy, nucleic acidamplification, gene array, protein array, mass spectrometry, patchclamp, 2-dimensional gel electrophoresis, differential display gelelectrophoresis, microsphere-based multiplex protein assays, ELISA, andlabel-free cellular assays to determine the activation levels of theplurality of intracellular activatable elements in single cells. In someembodiments, the determining step comprises the use of flow cytometry.In some embodiments, the classifying of single cells is further based onthe presence or absence of one or more cell surface markers,intracellular markers, or combinations thereof.

In another aspect, the invention provides a method of detecting thepresence or absence of disease-associated cells in an individual who hasreceived treatment comprising: subjecting a plurality of cells in asample from said individual to a modulator; determining the response ofsingle cells in the plurality of cells to said modulator; anddetermining the presence or absence of the disease-associated cellsbased on the response. In some embodiments, the method further comprisesdetermining the status of the individual based on said presence orabsence of disease-associated cells. In some embodiments, the diseaseassociated cells are rare cells.

In some embodiments, the response to the modulator comprises determiningthe activation level of an intracellular activatable element in saidsingle cells. In some embodiments, the method further comprises dividingthe sample into a plurality of subsamples, and subjecting each subsampleto a different modulator.

In some embodiments, the invention provides a method of detecting theminimal residual status of a disease in an individual who has receivedtreatment comprising subjecting a plurality of cells in a sample from anindividual to a modulator; determining the activation levels of aplurality of intracellular activatable elements in single cells inresponse to the modulator by a process comprising the binding of aplurality of binding elements which are specific to a particularactivation state of a particular activatable element, wherein the singlecells are placed into one or more classes based on said response to saidmodulator or modulators; determining the presence or absence of saiddisease-associated cells based on the response, wherein determining thepresence or absence of the disease-associated cells comprisesquantitative analysis of the one or more classes; and determining theminimal residual status of a disease, wherein the minimal residualstatus is based on the presence or absence of a small number of thedisease-associated cells. The minimal residual status refers to thenumber of disease-associated cells that remain in the individual duringtreatment or after treatment when the individual is in remission. Insome embodiments, the minimal residual status of a disease in anindividual is used to determine a health status in the individual.

In some embodiments, determining the response to the modulator comprisesdetermining the activation levels of a plurality of intracellularactivatable elements in said single cells. In some embodiments, theactivation level of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10(counting by whole numbers) intracellular activatable elements isdetermined. In some embodiments, the single cells are placed into one ormore classes based on said response to said modulator or modulators. Insome embodiments, the classes are predefined classes.

In some embodiments, the determining of the presence or absence of saiddisease-associated cells comprises quantitative analysis of classes. Insome embodiments, the classes are predefined classes. In someembodiments, the quantitative analysis of classes comprises determiningwhether or not said number of said cells in one or more of said classesis greater than, less than, or equal to a threshold number. In someembodiments, the threshold number is about 0, 1, 5, 10, 50, 100, 500,1000, 10,000, 100,000, or 1,000,000. In some embodiments, the methodcomprises determining whether or not said number of cells in a class isgreater than the threshold number 0.

In some embodiments, the method further comprises the determination ofthe ratio of the number of cells in one or more particular class orclasses of cells to the number of cells in one or more other class orclasses of cells, or to the total number of cells in the sample or afraction of the sample. In some embodiments, detecting the presence orabsence of disease-associated cells is determined by a processcomprising determining whether or not said ratio is greater than, lessthan, or equal to a threshold number. In some embodiments, the thresholdratio, expressed as a percentage, is about 0%, 0.0000001%, 0.000001%,0.00001%, 0.0001%, 001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, 5.0%,10%, 20%, 40%, 60%, 80%, 90%, 95%, or 100%.

In some embodiments, the quantitative analysis is performed on aplurality of samples from said individual. In some embodiments, theplurality of samples comprises samples from different locations in theindividual, samples taken at different times from the individual,samples treated in different ways prior to determining the activationlevel, or a combination thereof. In some embodiments, the plurality ofsamples comprises a series of samples taken from the individual atdifferent times.

In some embodiments, the method further comprises determining the rateof change in the number of cells in one or more of said classes, ordetermining the rate of change of the ratio of the number of cells inone or more particular class or classes of cells to the number of cellsin one or more other class or classes of cells, or to the total numberof cells in the sample or a fraction of the sample.

In some embodiments, the method further comprises determining anappropriate course of treatment for said individual based on said statusof the individual. In some embodiments, the appropriate course oftreatment comprises watchful waiting, supportive therapy, initiating atherapy, not initiating a therapy, stopping, shortening, prolonging, ormodifying an existing therapy, adding an additional therapy to existingtherapy, or combinations of the foregoing.

In some embodiments, the individual has received treatment for amalignant disorder. In some embodiments, the malignant disorder is asolid tumor or a hematologic malignancy. In some embodiments, themalignant disorder is non-B cell lineage derived. In some embodiments,the non-B cell lineage derived malignant disorder is selected from thegroup consisting of Acute myeloid leukemia (AML), Chronic MyeloidLeukemia (CML), non-B cell Acute lymphocytic leukemia (ALL), non-B celllymphomas, myelodysplastic disorders, myeloproliferative disorders,myelofibroses, polycythemias, thrombocythemias, and non-B cell atypicalimmune lymphoproliferations. In some embodiments, the non-B cell lineagederived malignant disorder is AML.

In some embodiments, the malignant disorder is a B cell or B celllineage derived disorder. In some embodiments, the malignant disorder isa B-Cell or B cell lineage derived disorder is selected from the groupconsisting of Chronic Lymphocytic Leukemia (CLL), B cell lymphocytelineage leukemia, B cell lymphocyte lineage lymphoma, Multiple Myeloma,and plasma cell disorders. In some embodiments, the B-Cell or B celllineage derived disorder is CLL.

In some embodiments, the status is expressed as a likelihood of returnor progression of a condition, or likelihood of a new conditiondeveloping.

In some embodiments, the modulator is an activator or an inhibitor. Insome embodiments, the modulator is a growth factor, cytokine, adhesionmolecule modulator, hormone, small molecule, polynucleotide, antibody,natural compound, lactone, chemotherapeutic agent, immune modulator,carbohydrate, protease, ion, reactive oxygen species, or radiation. Insome embodiments, the modulator is a B cell receptor modulator. In someembodiments, the B cell receptor modulator is a B cell receptoractivator. In some embodiments, the B cell receptor activator is acrosslinker is selected from the group consisting of F(ab)₂ IgM, IgG,IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptorderived binding elements.

In some embodiments, the modulator is an inhibitor, and wherein saidinhibitor is an inhibitor of a cellular factor or a plurality of factorsthat participates in a signaling cascade in said cell. In someembodiments, the inhibitor is a phosphatase inhibitor. In someembodiments, the phosphatase inhibitor is selected from the groupconsisting of H₂O₂, siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole,Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadylsulfate, Sodium oxodiperoxo(1,10-phenanthroline)vanadate,bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate,Sodium Tartrate, Imidazole, Sodium Fluoride, β-Glycerophosphate, SodiumPyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen),mpV(pic), DMHV, Cypermethrin, Dephostatin, Okadaic Acid, NIPP-1,N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide,α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br,and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,phenyarsine oxide, Pyrrolidine Dithiocarbamate, and Aluminium fluoride.In some embodiments, the phosphatase inhibitor is H₂O₂.

In some embodiments, the method further comprises subjecting the cellsto a second modulator concurrently with the first modulator. In someembodiments, the modulators are a B cell receptor activator and aphosphatase inhibitor. In some embodiments, the modulators are F(ab)₂IgMor biotinylated F(ab)₂IgM and H₂O₂.

In some embodiments, the activation level is based on the activationstate selected from the group consisting of cleavage by extracellular orintracellular protease exposure, novel hetero-oligomer formation,glycosylation state, phosphorylation state, acetylation state,methylation state, biotinylation state, glutamylation state, glycylationstate, hydroxylation state, isomerization state, prenylation state,myristoylation state, lipoylation state, phosphopantetheinylation state,sulfation state, ISGylation state, nitrosylation state, palmitoylationstate, SUMOylation state, ubiquitination state, neddylation state,citrullination state, deamidation state, disulfide bond formation state,proteolytic cleavage state, translocation state, changes in proteinturnover, multi-protein complex state, oxidation state, multi-lipidcomplex, and biochemical changes in cell membrane. In some embodiments,the activation state is a phosphorylation state.

In some embodiments, the activatable element is selected from the groupconsisting of proteins, carbohydrates, lipids, nucleic acids andmetabolites. In some embodiments, the activatable element is a protein.In some embodiments, the protein is a protein subject to phosphorylationand/or dephosphorylation.

In some embodiments, the protein is selected from the group consistingof kinases, phosphatases, lipid signaling molecules, adaptor/scaffoldproteins, cytokines, cytokine regulators, ubiquitination enzymes,adhesion molecules, cytoskeletal proteins, heterotrimeric G proteins,small molecular weight GTPases, guanine nucleotide exchange factors,GTPase activating proteins, caspases, proteins involved in apoptosis,cell cycle regulators, molecular chaperones, metabolic enzymes,vesicular transport proteins, hydroxylases, isomerases, deacetylases,methylases, demethylases, tumor suppressor genes, proteases, ionchannels, molecular transporters, transcription factors/DNA bindingfactors, regulators of transcription, and regulators of translation.

In some embodiments, the protein is selected from the group consistingof HER receptors, PDGF receptors, Kit receptor, FGF receptors, Ephreceptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor,Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn,Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF,Mos, Lim kinase, ILK, Tpl, ALK, TGFβ receptors, BMP receptors, MEKKs,ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub,Myt 1, Wee1, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3,p90Rsks, p70S6Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs,MNKs, AMPKs, MELK, MARKs, Chk1, Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3,IKKs, Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinaseclass 1, class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM,ATR, Receptor protein tyrosine phosphatases (RPTPs), LAR phosphatase,CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinasephosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25phosphatases, Low molecular weight tyrosine phosphatase, Eyes absent(EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serinephosphatases, PP2A, PP2B, PP2C, PP1, PP5, inositol phosphatases, PTEN,SHIPs, myotubularins, phosphoinositide kinases, phopsholipases,prostaglandin synthases, 5-lipoxygenase, sphingosine kinases,sphingomyelinases, adaptor/scaffold proteins, Shc, Grb2, BLNK, LAT, Bcell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL,GAD, Nck, Grb2 associated binder (GAB), Fas associated death domain(FADD), TRADD, TRAF2, RIP, T-Cell leukemia family, IL-2, IL-4, IL-8,IL-6, interferon γ, interferon α, suppressors of cytokine signaling(SOCs), Cbl, SCF ubiquitination ligase complex, APC/C, adhesionmolecules, integrins, Immunoglobulin-like adhesion molecules, selectins,cadherins, catenins, focal adhesion kinase, p130CAS, fodrin, actin,paxillin, myosin, myosin binding proteins, tubulin, eg5/KSP, CENPs,β-adrenergic receptors, muscarinic receptors, adenylyl cyclasereceptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras, Ran,Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam, Sos, Dbl, PRK, TSC1,2,Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Caspase 3, Caspase 6,Caspase 7, Caspase 8, Caspase 9, Bcl-2, Mcl-1, Bcl-XL, Bcl-w, Bcl-B, Al,Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma, IAPs, XIAP,Smac, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D, Cyclin E, Cyclin A,Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecular chaperones, Hsp90s,Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAa Carboxylase, ATP citratelyase, nitric oxide synthase, caveolins, endosomal sorting complexrequired for transport (ESCRT) proteins, vesicular protein sorting(Vsps), hydroxylases, prolyl-hydroxylases PHD-1, 2 and 3, asparaginehydroxylase FIH transferases, Pin1 prolyl isomerase, topoisomerases,deacetylases, Histone deacetylases, sirtuins, histone acetylases,CBP/P300 family, MYST family, ATF2, DNA methyl transferases, HistoneH3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-1, p53, Hdm, PTEN,ubiquitin proteases, urokinase-type plasminogen activator (uPA) and uPAreceptor (uPAR) system, cathepsins, metalloproteinases, esterases,hydrolases, separase, potassium channels, sodium channels, multi-drugresistance proteins, P-Gycoprotein, nucleoside transporters, Ets, Elk,SMADs, Rel-A (p65-NFKB), CREB, NFAT, ATF-2, AFT, Myc, Fos, Sp1, Egr-1,T-bet, β-catenin, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1, β-catenin, FOXOSTAT1, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-1, HMGA, pS6, 4EPB-1,eIF4E-binding protein, RNA polymerase, initiation factors, andelongation factors. In some embodiments, the protein is selected fromthe group consisting of Erk, Syk, Zap70, Lyn, Btk, BLNK, Cbl, PLCγ2,Akt, RelA, p38, S6. In some embodiments, the protein is S6.

In some embodiments, the activation level is determined by a processcomprising the binding of a binding element which is specific to aparticular activation state of the particular activatable element. Insome embodiments, the binding element comprises a protein. In someembodiments, the protein is an antibody. In some embodiments, theantibody binds to a activatable element selected from the groupconsisting of kinases, phosphatases, adaptor/scaffold proteins,ubiquitination enzymes, adhesion molecules, contractile proteins,cytoskeletal proteins, heterotrimeric G proteins, small molecular weightGTPases, guanine nucleotide exchange factors, GTPase activatingproteins, caspases and proteins involved in apoptosis, ion channels,molecular transporters, molecular chaperones, metabolic enzymes,vesicular transport proteins, hydroxylases, isomerases, transferases,deacetylases, methylases, demethylases, proteases, esterases,hydrolases, DNA binding proteins and transcription factors.

In some embodiments, the antibody binds to an activatable elementselected from the group consisting of HER receptors, PDGF receptors, Kitreceptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors,Insulin receptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK,Jak1, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk,ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK,TGFβ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1, Wee1, Casein kinases,PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3, p90Rsks, p70S6Kinase, Prks,PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs,Chk1, Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3, IKKs, Cdks, Jnks, Erks,IKKs, GSK3a, GSK3β, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, Receptor proteintyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptortyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs),Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecularweight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases,Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C,PP1, PP5, inositol phosphatases, PTEN, SHIPs, myotubularins,phosphoinositide kinases, phopsholipases, prostaglandin synthases,5-lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffoldproteins, Shc, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP),SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nck, Grb2 associated binder(GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-Cellleukemia family, IL-2, IL-4, IL-8, IL-6, interferon γ, interferon α,suppressors of cytokine signaling (SOCs), Cbl, SCF ubiquitination ligasecomplex, APC/C, adhesion molecules, integrins, Immunoglobulin-likeadhesion molecules, selectins, cadherins, catenins, focal adhesionkinase, p130CAS, fodrin, actin, paxillin, myosin, myosin bindingproteins, tubulin, eg5/KSP, CENPs, β-adrenergic receptors, muscarinicreceptors, adenylyl cyclase receptors, small molecular weight GTPases,H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam,Sos, Dbl, PRK, TSC1,2, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2,Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Bcl-2, Mcl-1,Bcl-XL, Bcl-w, Bcl-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk,Noxa, Puma, IAPs, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D,Cyclin E, Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecularchaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAaCarboxylase, ATP citrate lyase, nitric oxide synthase, caveolins,endosomal sorting complex required for transport (ESCRT) proteins,vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylasesPHD-1, 2 and 3, asparagine hydroxylase FIH transferases, Pin1 prolylisomerase, topoisomerases, deacetylases, Histone deacetylases, sirtuins,histone acetylases, CBP/P300 family, MYST family, ATF2, DNA methyltransferases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-1,p53, Hdm, PTEN, ubiquitin proteases, urokinase-type plasminogenactivator (uPA) and uPA receptor (uPAR) system, cathepsins,metalloproteinases, esterases, hydrolases, separase, potassium channels,sodium channels, multi-drug resistance proteins, P-Gycoprotein,nucleoside transporters, Ets, Elk, SMADs, Rel-A (p65-NFKB), CREB, NFAT,ATF-2, AFT, Myc, Fos, Sp1, Egr-1, T-bet, β-catenin, HIFs, FOXOs, E2Fs,SRFs, TCFs, Egr-1, β-catenin, FOXO STAT1, STAT 3, STAT 4, STAT 5, STAT6, p53, WT-1, HMGA, pS6, 4EPB-1, eIF4E-binding protein, RNA polymerase,initiation factors, and elongation factors.

In some embodiments, the step of determining the activation levelcomprises the use of flow cytometry, immunofluorescence, confocalmicroscopy, immunohistochemistry, immunoelectronmicroscopy, nucleic acidamplification, gene array, protein array, mass spectrometry, patchclamp, 2-dimensional gel electrophoresis, differential display gelelectrophoresis, microsphere-based multiplex protein assays, ELISA, andlabel-free cellular assays to determine the activation level of one ormore intracellular activatable element in single cells. In someembodiments, the determining step comprises the use of flow cytometry.

In some embodiments, determining the presence or absence of thedisease-associated cells is further based on the presence or absence ofone or more cell surface markers, the presence or absence of one or moreintracellular markers, or a combination thereof.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a graph illustrating the change in the number of a predefinedclass of cells over time. Here, the cell number is increasing and by thesixth measurement has exceeded the threshold number.

FIG. 2 illustrates the detection and quantification of multiplepredefined classes of cells in a sample. 2A. Numerous predefined classescan be observed and quantified when multiple binding elements tointracellular activatable elements are employed, particularly ifphysical parameters like cell volume or density and additionalbiochemical information such as the expression level of cell surfacemarkers or nuclear antigens is employed. 2B Various comparisons can bemade between classes including taking the ratio of the cell numbersfound in particular classes.

FIG. 3 is a graph illustrating the change in the ratio of predefinedclasses over time. Here, the ratio has decreased over time and by thefourth measurement has dropped below the threshold number

FIG. 4 is a graph illustrating the rate of change in the cell number twodifferent predefined classes of cells over time. In one cell population,illustrated by the thick line, the rate of change in the cell populationis decreasing, while in the other population, illustrated by the thinline, the rate of change is increasing.

FIG. 5 shows identification of relevant subpopulations in BMMCs from MDSpatients. Myeloblasts, mature monocytes, nRBCs, and lymphocytes aregated based on CD45, CD235ab, CD71, CD34, CD33 and CD11b expression aswell as FSC and SSC profiles.

FIG. 6 shows identification of erythroid cells at differentdevelopmental stages from normal and MDS patient bone marrow based ontheir CD235ab and CD71 expression profiles.

FIG. 7 shows analysis of erythroid precursors in normal versus MDS bonemarrow. The results reveal a block of erythroid differentiation in MDS.

FIG. 8 shows STAT5 and STAT1 phosphorylation in rRBCs from normal andMDS patients in response to erythropoietin (EPO) stimulation. nRBCsubpopulation from MDS patients exhibits STAT5 phosphorylation inresponse to EPO stimulation.

FIG. 9 shows STAT5 and STAT1 phosphorylation in rRBCs from normal andMDS patients in response to interferon gamma (IFNγ) stimulation. nRBCsubpopulation from MDS patients exhibits STAT1 phosphorylation inresponse to IFNγ stimulation.

FIG. 10 shows a concentration dependent loss of CD34+ myeloblast cellsin healthy BMMCs in the presence of 5-Azacytidine.

FIG. 11 shows that Decitabine (Dacogen) does not affect the viability ofCD34+ myeloblast cells.

FIG. 12 shows a concentration dependent loss of CD34+ myeloblast cellsin healthy BMMCs in the presence of Vorinostat (Zolinza).

FIG. 13 shows CD45RA/RO/RB expression profiles of mature monocytes,myeloblasts, and lymphocytes

FIG. 14 shows CD45RA/RO/RB expression profiles of mature monocytes,myeloblasts, and lymphocytes from bone marrow of MDS patient 03.

FIG. 15 is a diagram showing the method of determining a status of anindividual at different stages. The method can be applied to anindividual before a diagnosis, an individual undergoing a treatment, oran individual undergoing remission or having a relapse.

FIG. 16 shows p-Stat5 and p-Stat1 levels in myeloid cells from a patientat the time of diagnosis or at relapse.

FIG. 17 shows p-AKT and p-S6 levels in myeloid cells from a patient atthe time of diagnosis and post induction therapy.

FIG. 18 shows p-AKT and p-S6 levels in CD33, CD11b⁻, CD34⁺ cells in anAML patient.

FIG. 19 shows the frequency of p-AKT/pS6 myeloid cells responsive to SCFin different AML patients.

DETAILED DESCRIPTION OF THE INVENTION

The present invention incorporates information disclosed in otherapplications and texts. The following patent and other publications arehereby incorporated by reference in their entireties: Haskell et al,Cancer Treatment, 5^(th) Ed., W.B. Saunders and Co., 2001; Alberts etal., The Cell, 4^(th) Ed., Garland Science, 2002; Vogelstein andKinzler, The Genetic Basis of Human Cancer, 2d Ed., McGraw Hill, 2002;Michael, Biochemical Pathways, John Wiley and Sons, 1999; Immunobiology,Janeway et al. 7^(th) Ed., Garland, and Leroith and Bondy, GrowthFactors and Cytokines in Health and Disease, A Multi Volume Treatise,Volumes 1A and 1B, Growth Factors, 1996. Patent applications that arealso incorporated by reference include U.S. Ser. Nos. 10/193,462;11/655,785; 11/655,789; 10/346,620; 11/655,821; 10/898,734; and11/338,957. Some commercial reagents, protocols, software andinstruments that are useful in some embodiments of the present inventionare available at the Becton Dickinson Websitehttp://www.bdbiosciences.com/features/products/, and the Beckman Coulterwebsite, http://www.beckmancoulter.com/Default.asp?bhfv=7. Relevantarticles include High-content single-cell drug screening withphosphospecific flow cytometry, Krutzik et al., Nature Chemical Biology,23 Dec. 2007; Irish et al., Flt3 Y591 duplication and Bcl-2 overexpression are detected in acute myeloid leukemia cells with high levelsof phosphorylated wild-type p53, Neoplasia, 2007, and Irish et al.,Single cell profiling of potentiated phospho-protein networks in cancercells, Cell, Vol. 118, 1-20 Jul. 23, 2004; Schulz, K. R., et al.,Single-cell phospho-protein analysis by flow cytometry, Curr ProtocImmunol, 2007, 78:8 8.17.1-20; Krutzik, P. O., et al., Coordinateanalysis of murine immune cell surface markers and intracellularphosphoproteins by flow cytometry, J. Immunol. 2005 Aug. 15;175(4):2357-65; Krutzik, P. O., et al., Characterization of the murineimmunological signaling network with phosphospecific flow cytometry, J.Immunol. 2005 Aug. 15; 175(4):2366-73; and Krutzik, P. O. and Nolan, G.P., Intracellular phospho-protein staining techniques for flowcytometry: monitoring single cell signaling events, Cytometry A. 2003October; 55(2):61-70. Experimental and process protocols and otherhelpful information can be found at http:/proteomices.stanford.edu.

One embodiment of the invention is directed to methods for determiningthe status of an individual by determining the activation level ofindividual cells in one or more samples obtained from the individual.Typically, the status of an individual will be the health status, butany type of status can be determined if it can be correlated to thestatus of single cells in a sample from the individual. In someembodiments, the invention provides methods for determining the statusof an individual by detecting one or more rare cell populations. Thus,the invention provides methods for the determination of the status of anindividual by analyzing one or more rare populations of cells, usuallynot detectable by other methods known in the art, while keeping a highlevel of statistical significance in the determination. In someembodiments, the invention provides methods for early determination ofthe individual status. For example, in the case of diagnosis of apathological state the invention provides for early diagnosis of thepathological state, e.g., before the individual presents any symptoms.

In some embodiments the status of the individual is the minimal statusof a pathological state. Thus, in some embodiments, the invention isdirected to determining the minimal status of a pathological state in anindividual by determining the activation level of individual cells inone or more samples obtained from the individual. The “minimal status”of a pathological state as used herein refers to the minimum number ofcells indicative of a pathological state. In some embodiments, theminimal status of a pathological state in the minimum numbers of cellsrequired to make a diagnosis for the pathological state. In certaininstances, the finding of 0 cells associated with a pathological statemay be determinative as to minimal status of a pathological state. Forexample, the finding of 0 cells associated with a pathological stateprovides evidence that the individual does not have the pathologicalstate or has not experienced a recurrence. In some embodiments, thepresence of 1 cell associated with a pathological state may bedeterminative of an individual's status. In this case, the thresholdnumber is 0, and finding even a single cell (more than zero) isindicative of the minimal status of the pathological state. For example,the finding of 1 cell that is associated with a highly malignant cancerphenotype indicates that the in the case of cancer, the disease processhas begun, but may be yet to manifest disease symptoms. In an individualwho has been treated for the pathological condition, the detection ofcells associated with the pathological state indicates that treatment isincomplete. In other instances, a finding of a number higher than athreshold of cells associated with a pathological state may bedeterminative of an individual's status, wherein the threshold in theminimum number of cells required to make a determination of theindividual's status. For example, a finding of equal or higher that 10⁻⁴cells associated with a cancer phenotype may indicate that theindividual is at risk of having a relapse, whereas a finding of lessthan 10⁻⁴ cells may indicate that the individual is at very low risk ofrelapse.

In some embodiments, the status of the single cells in the sample isdetermined, e.g., by determining the status of one or more activatableelements in the cells. The activatable elements may be proteins; in someembodiments, the activatable elements are phosphoproteins. The cells maythen be classified into one or more classes, depending on the activationlevel of the one or more activatable elements, and a quantitativeanalysis is performed on the number of cells in one or more of theclasses. In some embodiments, cells are treated with a modulator beforetheir status is determined. See U.S. Ser. No. 10/898,734.

In some embodiments, the health status of an individual places theindividual along a health continuum that typically runs from a healthystate to one or more pre-pathologic states, and finally to a pathologicstate. In some instances, the health continuum may run from a healthystate to a pathological state without an intervening pre-pathologicstate. The health continuum may also comprise a partial continuum of theaforementioned states or a portion of one state. The health continuummay be related to the general health status of an individual, an organor organ system or the individual component tissues of an organ.Additionally, the health continuum may be specific for a family ofrelated diseases or disorder, a particular disease or disorder or asubtype of a disease or disorder. See Haskell et al, Cancer Treatment,5^(th) Ed., W.B. Saunders and Co., 2001

Diseases, disorders, and conditions encompassed by a health continuumcan include an immunologic, malignant, or proliferative disease ordisorder, or one that has characteristics from a combination of thesedisorders. See Immunobiology, Janeway et al. 7^(th) Ed., Garland.Diseases that are especially likely to progress along a continuum fromhealth to prepathological to pathological are cancers, which typicallyrequire a series of genetic changes in order to progress to malignancy.Cancers that are especially amenable to evaluation and interventioninclude those that are associated with the blood, i.e., hematologicmalignancies, because blood is easily sampled and processed. An exampleof a malignancy that progresses along such a continuum, which serves asan example of disorders that may be evaluated by the methods of theinvention, is AML. AML can be preceded by a prepathological stage,myelodysplastic disorder (MDS). The methods of the invention allowmonitoring of an individual at a series of time points to determinewhere on the continuum from healthy, through MDS (prepathological) toAML (pathological), the individual is situated. See Haskell et al,Cancer Treatment, 5^(th) Ed., W.B. Saunders and Co., 2001

Knowing the health status of an individual allows for the diagnosis,prognosis, choice or modification of treatment, and/or monitoring of adisease, disorder, or condition. Through the determination of the healthstatus of an individual, a health care practitioner can assess whetherthe individual is in the normal range for a particular condition orwhether the individual has a pre-pathological or pathological conditionwarranting monitoring and/or treatment. This type of methodology can beparticularly important with diseases or conditions where an individualis asymptomatic and appears normal. This is often the case with manytypes of cancer, which may be asymptomatic for months or years andwhich, at the time symptoms appear, may be much less amenable totreatment than if they had been detected earlier.

The determination of the health status may also indicate response of anindividual to treatment for a condition. Such information allows forongoing monitoring of the condition and/or additional treatment. In oneembodiment, the invention provides for the detection of the presence ofdisease-associated cells or the absence or reduction of cells necessaryfor normal physiology in an individual that is being treated, or waspreviously treated, for the disease or condition. The disease-associatedcells may be cancerous and may be present at sufficiently low numbers soas not to cause overt symptoms or be detectable by imaging modalities,clinical exam, or routine clinical screening labs e.g. complete bloodcount. In some embodiments, the invention provides for the detection ofa slight reduction in a normal cell population that precedes oraccompanies a disease process. In some embodiments the disease processcomprises a malignancy.

In some embodiments, the determination of the health status of anindividual may be used to ascertain whether a previous condition ortreatment has induced a new pre-pathological or pathological conditionthat requires monitoring and/or treatment. For example, treatment formany forms of cancers (e.g. lymphomas and childhood leukemias) caninduce certain adult leukemias, and the methods of the present inventionallow for the early detection and treatment of such leukemias.

In another embodiment, the status of an individual can indicate anindividual's predicted or actual response to treatment for apre-pathological or pathological condition. This predictive informationcan be obtained through the analysis of the same, additional ordifferent parameters than those used to place the individual along thehealth continuum. Predictive information may be used to determine thebest therapy for an individual, which may include the determination thatthe best therapy for a patient is supportive care.

In a further embodiment, the status of an individual may indicate anindividual's immunologic status and may reflect a general immunologicstatus, an organ or tissue specific status, or a disease related status.

Samples and Sampling

The methods involve analysis of one or more samples from an individual.An individual is any multicellular organism; in some embodiments, theindividual is an animal, e.g., a mammal. In some embodiments, theindividual is a human.

The sample may be any suitable type that allows for the analysis ofsingle cells. Samples may be obtained once or multiple times from anindividual. Multiple samples may be obtained from different locations inthe individual (e.g., blood samples, bone marrow samples and/or lymphnode samples), at different times from the individual (e.g., a series ofsamples taken to monitor response to treatment or to monitor for returnof a pathological condition), or any combination thereof. These andother possible sampling combinations based on the sample type, locationand time of sampling allows for the detection of the presence ofpre-pathological or pathological cells, the measurement treatmentresponse and also the monitoring for disease.

When samples are obtained as a series, e.g., a series of blood samplesobtained after treatment, the samples may be obtained at fixedintervals, at intervals determined by the status of the most recentsample or samples or by other characteristics of the individual, or somecombination thereof. For example, samples may be obtained at intervalsof approximately 1, 2, 3, or 4 weeks, at intervals of approximately 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, at intervals of approximately1, 2, 3, 4, 5, or more than 5 years, or some combination thereof. Itwill be appreciated that an interval may not be exact, according to anindividual's availability for sampling and the availability of samplingfacilities, thus approximate intervals corresponding to an intendedinterval scheme are encompassed by the invention. As an example, anindividual who has undergone treatment for a cancer may be sampled(e.g., by blood draw) relatively frequently (e.g., every month or everythree months) for the first six months to a year after treatment, then,if no abnormality is found, less frequently (e.g., at times between sixmonths and a year) thereafter. If, however, any abnormalities or othercircumstances are found in any of the intervening times, or during thesampling, sampling intervals may be modified.

Generally, the most easily obtained samples are fluid samples. Fluidsamples include normal and pathologic bodily fluids and aspirates ofthose fluids. Fluid samples also comprise rinses of organs and cavities(lavage and perfusions). Bodily fluids include whole blood, bone marrowaspirate, synovial fluid, cerebrospinal fluid, saliva, sweat, tears,semen, sputum, mucus, menstrual blood, breast milk, urine, lymphaticfluid, amniotic fluid, placental fluid and effusions such as cardiaceffusion, joint effusion, pleural effusion, and peritoneal cavityeffusion (ascites). Rinses can be obtained from numerous organs, bodycavities, passage ways, ducts and glands. Sites that can be rinsedinclude lungs (bronchial lavage), stomach (gastric lavage),gastrointestinal track (gastrointestinal lavage), colon (colonicsavage), vagina, bladder (bladder irrigation), breast duct (ductalsavage), oral, nasal, sinus cavities, and peritoneal cavity (peritonealcavity perfusion). In some embodiments the sample or samples is blood.

Solid tissue samples may also be used, either alone or in conjunctionwith fluid samples. Solid samples may be derived from individuals by anymethod known in the art including surgical specimens, biopsies, andtissue scrapings, including cheek scrapings. Surgical specimens includesamples obtained during exploratory, cosmetic, reconstructive, ortherapeutic surgery. Biopsy specimens can be obtained through numerousmethods including bite, brush, cone, core, cytological, aspiration,endoscopic, excisional, exploratory, fine needle aspiration, incisional,percutaneous, punch, stereotactic, and surface biopsy.

In some embodiments, the sample is a blood sample. In some embodiments,the sample is a bone marrow sample. In some embodiments, the sample is alymph node sample. In some embodiments, the sample is cerebrospinalfluid. In some embodiments, combinations of one or more of a blood, bonemarrow, cerebrospinal fluid, and lymph node sample are used.

In one embodiment, a sample may be obtained from an apparently healthyindividual during a routine checkup and analyzed so as to provide anassessment of the individual's general health status. In anotherembodiment, a sample may be taken to screen for commonly occurringdiseases. Such screening may encompass testing for a single disease, afamily of related diseases or a general screening for multiple,unrelated diseases. Screening can be performed weekly, bi-weekly,monthly, bimonthly, every several months, annually, or in several yearintervals and may replace or complement existing screening modalities.

In another embodiment, an individual with a known increased probabilityof disease occurrence may be monitored regularly to detect for theappearance of a particular disease or class of diseases. An increasedprobability of disease occurrence can be based on familial association,age, previous genetic testing results, or occupational, environmental ortherapeutic exposure to disease causing agents. Breast and ovariancancer related to inherited mutations in the genes BRCA1 and BRCA2 areexamples of diseases with a familial association wherein susceptibleindividuals can be identified through genetic testing. Another exampleis the presence of inherited mutations in the adenomatous polyposis coligene predisposing individuals to colorectal cancer. Examples ofenvironmental or therapeutic exposure include individuals occupationallyexposed to benzene that have increased risk for the development ofvarious forms of leukemia, and individuals therapeutically exposed toalkylating agents for the treatment of earlier malignancies. Individualswith increased risk for specific diseases can be monitored regularly forthe first signs of an appearance of an abnormal cell population.Monitoring can be performed weekly, bi-weekly, monthly, bimonthly, everyseveral months, annually, or in several year intervals, or anycombination thereof. Monitoring may replace or complement existingscreening modalities. Through routine monitoring, early detection of thepresence of disease causative or associated cells may result inincreased treatment options including treatments with lower toxicity andincreased chance of disease control or cure.

In a further embodiment, testing can be performed to confirm or refutethe presence of a suspected genetic or physiologic abnormalityassociated with increased risk of disease. Such testing methodologiescan replace other confirmatory techniques like cytogenetic analysis orfluorescent in situ histochemistry (FISH). In still another embodiment,testing can be performed to confirm or refute a diagnosis of apre-pathological or pathological condition.

In instances where an individual has a known pre-pathologic orpathologic condition, a plurality of single cells from the appropriatelocation can be sample and analyzed to predict the response of theindividual to available treatment options. In one embodiment, anindividual treated with the intent to reduce in number or ablate cellsthat are causative or associated with a pre-pathological or pathologicalcondition can be monitored to assess the decrease in such cells overtime. A reduction in causative or associated cells may or may not beassociated with the disappearance or lessening of disease symptoms. Ifthe anticipated decrease in cell number does not occur, furthertreatment with the same or a different treatment regiment may bewarranted.

In another embodiment, an individual treated to reverse or arrest theprogression of a pre-pathological condition can be monitored to assessthe reversion rate or percentage of cells arrested at thepre-pathological status point. If the anticipated reversion rate is notseen or cells do not arrest at the desired pre-pathological status pointfurther treatment with the same or a different treatment regiment can beconsidered.

In a further embodiment, cells of an individual can be analyzed to seeif treatment with a differentiating agent has pushed a cell type along aspecific tissue lineage and to terminally differentiate with subsequentloss of proliferative or renewal capacity. Such treatment may be usedpreventively to keep the number of dedifferentiated cells associatedwith disease at a low level thereby preventing the development of overtdisease. Alternatively, such treatment may be used in regenerativemedicine to coax or direct pluripotent or multipotent stem cells down adesired tissue or organ specific lineage and thereby accelerate orimprove the healing process.

Individuals may also be monitored for the appearance or increase in cellnumber of another predefined class or classes of cells that areassociated with a good prognosis. If a beneficial, predefined class ofcells is observed, measures can be taken to further increase theirnumbers, such as the administration of growth factors. Alternatively,individuals may be monitored for the appearance or increase in cellnumber of another predefined class or classes of cells associated with apoor prognosis. In such a situation, renewed therapy can be consideredincluding continuing, modifying the present therapy or initiatinganother type of therapy.

In these embodiments, one or more samples may be taken from theindividual, and subjected to a modulator, as described herein. In someembodiments, the sample is divided into subsamples that are eachsubjected to a different modulator. After treatment with the modulator,single cells in the sample or subsample are analyzed to determine theiractivation level(s). Any suitable form of analysis that allows adetermination of cell activation level(s) may be used. In someembodiments, the analysis includes the determination of the activationlevel of an intracellular element, e.g., a protein. In some embodiments,the analysis includes the determination of the activation level of anactivatable element, e.g., an intracellular activatable element such asa protein, e.g., a phosphoprotein. Determination of the status may beachieved by the use of activation state-specific binding elements, suchas antibodies, as described herein. A plurality of activatable elementsmay be examined. Single cells may be placed into predefined classes, andthe status of the individual determined based on the classes into whichcells are categorized. In some embodiments, a quantitative analysis ofthe number of cells in one or more classes is performed to determine thestatus of the individual.

Certain fluid samples can be analyzed in their native state with orwithout the addition of a diluent or buffer. Alternatively, fluidsamples may be further processed to obtain enriched or purified cellpopulations prior to analysis. Numerous enrichment and purificationmethodologies for bodily fluids are known in the art. A common method toseparate cells from plasma in whole blood is through centrifugationusing heparinized tubes. By incorporating a density gradient, furtherseparation of the lymphocytes from the red blood cells can be achieved.A variety of density gradient media are known in the art includingsucrose, dextran, bovine serum albumin (BSA), FICOLL diatrizoate(Pharmacia), FICOLL metrizoate (Nycomed), PERCOLL (Pharmacia),metrizamide, and heavy salts such as cesium chloride. Alternatively, redblood cells can be removed through lysis with an agent such as ammoniumchloride prior to centrifugation.

Whole blood can also be applied to filters that are engineered tocontain pore sizes that select for the desired cell type or class. Forexample, rare pathogenic cells can be filtered out of diluted, wholeblood following the lysis of red blood cells by using filters with poresizes between 5 to 10 μm, as disclosed in U.S. patent application Ser.No. 09/790,673. Alternatively, whole blood can be separated into itsconstituent cells based on size, shape, deformability or surfacereceptors or surface antigens by the use of a microfluidic device asdisclosed in U.S. patent application Ser. No. 10/529,453.

Select cell populations can also be enriched for or isolated from wholeblood through positive or negative selection based on the binding ofantibodies or other entities that recognize cell surface or cytoplasmicconstituents. For example, U.S. Pat. No. 6,190,870 to Schmitz et al.discloses the enrichment of tumor cells from peripheral blood bymagnetic sorting of tumor cells that are magnetically labeled withantibodies directed to tissue specific antigens.

Solid tissue samples may require the disruption of the extracellularmatrix or tissue stroma and the release of single cells for analysis.Various techniques are known in the art including enzymatic andmechanical degradation employed separately or in combination. An exampleof enzymatic dissociation using collagenase and protease can be found inWolters G H J et al. An analysis of the role of collagenase and proteasein the enzymatic dissociation of the rat pancrease for islet isolation.Diabetologia 35:735-742, 1992. Examples of mechanical dissociation canbe found in Singh, N P. Technical Note: A rapid method for thepreparation of single-cell suspensions from solid tissues. Cytometry31:229-232 (1998). Alternately, single cells may be removed from solidtissue through microdissection including laser capture microdissectionas disclosed in Laser Capture Microdissection, Emmert-Buck, M. R. et al.Science, 274(8):998-1001, 1996.

In some embodiments, single cells can be analyzed within a tissuesample, such as a tissue section or slice, without requiring the releaseof individual cells before determining step is performed.

Modulators

In some embodiments the sample may be treated with at least onemodulator. Such treatment can yield information regarding the state ofsingle cells that is useful in determining the status of the individual.In some embodiments, the sample is divided into subsamples which areeach treated with a different modulator. A modulator causes modificationof one or more activatable elements of a cell (e.g., activation ordeactivation), a change in expression of an element, or the localizationof an element, generally as part of a signaling pathway, in at least onetype of cell. A modulator may be an activator or an inhibitor—e.g., amodulator may activate one or more activatable elements in one or morecellular signaling pathways, or inhibit one or more activatable elementsin one or more cellular pathways. See U.S. Ser. Nos. 10/193,462;11/655,785; 11/655,789; 10/346,620; 11/655,821; 10/898,734; and11/338,957.

Cells can be treated with a modulator as a single pulse, or withsequential pulses. With sequential treatment, a modulator can be used atthe same concentration and duration of exposure or at differentconcentrations and exposure. In some embodiments, cells are treated withtwo modulators. In some embodiments, cells are treated with 3, 4, 5, 6,7, 8, 9, 10, or more modulators. These modulators can both beactivators, inhibitors, or one can be an activator and the other aninhibitor. Treatment can consist of simultaneous or sequential exposureto a combination of modulators. As an illustrative example, a cell canbe treated simultaneously with a B cell receptor activator such asF(ab)₂IgM and a phosphatase inhibitor like H₂O₂.

Modulation can be performed in a variety of environments. In someembodiments, cells are exposed to a modulator immediately aftercollection. In some embodiments where there is a mixed population ofcells, purification of cells is performed after modulation. In someembodiments, whole blood is collected to which is added a modulator. Insome embodiments, cells are modulated after processing for single cellsor purified fractions of single cells. As an illustrative example, wholeblood can be collected and processed for an enriched fraction oflymphocytes that is then exposed to a modulator.

In some embodiments, cells are cultured post collection in a suitablemedia before exposure to a modulator. In some embodiments, the media isa growth media. In some embodiments, the growth media is a complex mediathat may include serum. In some embodiments, the growth media comprisesserum. In some embodiments, the serum is selected from the groupconsisting of fetal bovine serum, bovine serum, human serum, porcineserum, horse serum, and goat serum. In some embodiments, the serum levelranges from 0.0001% to 30%. In some embodiments, the growth media is achemically defined minimal media and is without serum. In someembodiments, cells are cultured in a differentiating media.

Modulators include chemical and biological entities, and physical orenvironmental stimuli. Modulators can act extracellularly orintracellularly. Chemical and biological modulators include growthfactors, cytokines, neurotransmitters, adhesion molecules, hormones,small molecules, inorganic compounds, polynucleotides, antibodies,natural compounds, lectins, lactones, chemotherapeutic agents,biological response modifiers, carbohydrate, proteases and freeradicals. Modulators include complex and undefined biologic compositionsthat may comprise cellular or botanical extracts, cellular or glandularsecretions, physiologic fluids such as serum, amniotic fluid, or venom.Physical and environmental stimuli include electromagnetic, ultraviolet,infrared or particulate radiation, redox potential and pH, the presenceor absences of nutrients, changes in temperature, changes in oxygenpartial pressure, changes in ion concentrations and the application ofoxidative stress. Modulators can be endogenous or exogenous and mayproduce different effects depending on the concentration and duration ofexposure to the single cells or whether they are used in combination orsequentially with other modulators. Modulators can act directly on theactivatable elements or indirectly through the interaction with one ormore intermediary biomolecule. Indirect modulation includes alterationsof gene expression wherein the expressed gene product is the activatableelement or is a modulator of the activatable element.

Modulators that are activators include ligands for cell surfacereceptors such as hormones, growth factors and cytokines. Otherextracellular activators include antibodies or molecular bindingentities that recognize cell surface markers or receptors including Bcell receptor complex, B cell co-receptor complex or surfaceimmunoglobulins. In one embodiment, cell surface markers, receptors orimmunoglobulins are crosslinked by the activators. In a furtherembodiment, the crosslinking activator is a polyclonal IgM antibody, amonoclonal IgM antibody, F(ab)₂ IgM, biotinylated F(ab)₂ IgM,biotinylated polyclonal anti-IgM, or biotinylated monoclonal anti-IgM.In some embodiments, the modulator is a B cell receptor modulator. Insome embodiments, the B cell receptor modulator is a B cell receptoractivator.

An example of B cell receptor activator is a cross-linker of the B cellreceptor complex or the B-cell co-receptor complex. In some embodiments,cross-linker is an antibody or molecular binding entity. In someembodiments, the cross-linker is an antibody. In some embodiments, theantibody is a multivalent antibody. In some embodiments, the antibody isa monovalent, bivalent, or multivalent antibody made more multivalent byattachment to a solid surface or tethered on a nanoparticle surface toincrease the local valency of the epitope binding domain.

In some embodiments, the cross-linker is a molecular binding entity. Insome embodiments, the molecular binding entity acts upon or binds the Bcell receptor complex via carbohydrates or an epitope in the complex. Insome embodiments, the molecular is a monovalent, bivalent, ormultivalent is made more multivalent by attachment to a solid surface ortethered on a nanoparticle surface to increase the local valency of theepitope binding domain.

In some embodiments, the cross-linking of the B cell receptor complex orthe B-cell co-receptor complex comprises binding of an antibody ormolecular binding entity to the cell and then causing its crosslinkingvia interaction of the cell with a solid surface that causescrosslinking of the BCR complex via antibody or molecular bindingentity.

In some embodiments, the crosslinker is F(ab)₂ IgM, IgG, IgD, polyclonalBCR antibodies, monoclonal BCR antibodies, or Fc receptor derivedbinding elements. In some embodiments, the Ig is derived from a speciesselected from the group consisting of mouse, goat, rabbit, pig, rat,horse, cow, shark, chicken, or llama. In some embodiments, thecrosslinker is F(ab)₂ IgM, Polyclonal anti-IgM, Monoclonal anti-IgM,Biotinylated F(ab)₂ IgCM, Biotinylated Polyclonal anti-IgM, orBiotinylated Monoclonal anti-IgM.

Inhibitory modulators include inhibitors of a cellular factor or aplurality of cellular factors that participate in a cell signalingpathway. Inhibitors include a phosphatase inhibitor, such as H₂O₂,siRNA, miRNA, cantharidin, (−)-p-Bromotetramisole, Microcystin LR,Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodiumoxodiperoxo(1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV),Sodium Molybdate, Sodium Permolybdate, Sodium Tartrate, Imidazole,Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate,Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin,Dephostatin, Okadaic Acid, NIPP-1,N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide,α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br,and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,phenyarsine oxide, Pyrrolidine Dithiocarbamate, or Aluminium fluoride.In some embodiments, the modulator is the phosphatase inhibitor H₂O₂.

In some embodiments, the methods of the invention provides for the useof more than one modulator. In some embodiments, the methods of theinvention utilize a B cell receptor activator and a phosphataseinhibitor. In some embodiments, the methods of the invention utilizeF(ab)₂IgM or biotinylated F(ab)₂IgM and H₂O₂.

Other modulators suitable for use in the invention are described in U.S.patent application Ser. Nos. 10/193,462; 10/898,734; 10/346,620; and11/338,957, all of which are incorporated herein by reference in theirentirety.

Determination of Cell Status

After treatment with one or more modulators, if used, in someembodiments the sample is analyzed to find the activation level of anactivatable element in single cells. Any suitable analysis that allowsdetermination of the activation level of an activatable element withinsingle cells, which provides information useful for determining thestatus of the individual from whom the sample was taken, may be used.Examples include flow cytometry, immunohistochemistry, immunofluorescenthistochemistry with or without confocal microscopy,immunoelectronmicroscopy, nucleic acid amplification, gene array,protein array, mass spectrometry, patch clamp, 2-dimensional gelelectrophoresis, differential display gel electrophoresis,microsphere-based multiplex protein assays, ELISA, Inductively CoupledPlasma Mass Spectrometer (ICP-MS) and label-free cellular assays.Additional information for the further discrimination between singlecells can be obtained by many methods known in the art including thedetermination of the presence of absence of extracellular and/orintracellular markers, the presence of metabolites, gene expressionprofiles, DNA sequence analysis, and karyotyping.

Activatable Elements

In some embodiments, the activation level of one or more activatableelements in single cells in the sample determined. Cellular constituentsthat may include activatable elements include without limitationproteins, carbohydrates, lipids, nucleic acids and metabolites. Theactivatable element may be a portion of the cellular constituent, forexample, an amino acid residue in a protein that may undergophosphorylation, or it may be the cellular constituent itself, forexample, a protein that is activated by translocation, change inconformation (due to, e.g., change in pH or ion concentration), byproteolytic cleavage, and the like. Upon activation, a change occurs tothe activatable element, such as covalent modification of theactivatable element (e.g., binding of a molecule or group to theactivatable element, such as phosphorylation) or a conformationalchange. Such changes generally contribute to changes in particularbiological, biochemical, or physical properties of the cellularconstituent that contains the activatable element. The state of thecellular constituent that contains the activatable element is determinedto some degree, though not necessarily completely, by the state of aparticular activatable element of the cellular constituent. For example,a protein may have multiple activatable elements, and the particularactivation states of these elements may overall determine the activationstate of the protein; the state of a single activatable element is notnecessarily determinative. Additional factors, such as the binding ofother proteins, pH, ion concentration, interaction with other cellularconstituents, and the like, can also affect the state of the cellularconstituent.

In some embodiments, the activation levels of a plurality ofintracellular activatable elements in single cells are determined. Insome embodiments, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than10 intracellular activatable elements are determined.

Activation states of activatable elements may result from chemicaladditions or modifications of biomolecules and include biochemicalprocesses such as glycosylation, phosphorylation, acetylation,methylation, biotinylation, glutamylation, glycylation, hydroxylation,isomerization, prenylation, myristoylation, lipoylation,phosphopantetheinylation, sulfation, ISGylation, nitrosylation,palmitoylation, SUMOylation, ubiquitination, neddylation,citrullination, amidation, and disulfide bond formation, disulfide bondreduction. Other possible chemical additions or modifications ofbiomolecules include the formation of protein carbonyls, directmodifications of protein side chains, such as o-tyrosine, chloro-,nitrotyrosine, and dityrosine, and protein adducts derived fromreactions with carbohydrate and lipid derivatives. Other modificationsmay be non-covalent, such as binding of a ligand or binding of anallosteric modulator.

Examples of proteins that may include activatable elements include, butare not limited to kinases, phosphatases, lipid signaling molecules,adaptor/scaffold proteins, cytokines, cytokine regulators,ubiquitination enzymes, adhesion molecules, cytoskeletal/contractileproteins, heterotrimeric G proteins, small molecular weight GTPases,guanine nucleotide exchange factors, GTPase activating proteins,caspases, proteins involved in apoptosis, cell cycle regulators,molecular chaperones, metabolic enzymes, vesicular transport proteins,hydroxylases, isomerases, deacetylases, methylases, demethylases, tumorsuppressor genes, proteases, ion channels, molecular transporters,transcription factors/DNA binding factors, regulators of transcription,and regulators of translation. Examples of activatable elements,activation states and methods of determining the activation level ofactivatable elements are described in US Publication Number 20060073474entitled “Methods and compositions for detecting the activation state ofmultiple proteins in single cells” and US Publication Number 20050112700entitled “Methods and compositions for risk stratification” the contentof which are incorporate here by reference.

In some embodiments, the protein is selected from the group consistingof HER receptors, PDGF receptors, Kit receptor, FGF receptors, Ephreceptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor,Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn,Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF,Mos, Lim kinase, ILK, Tpl, ALK, TGFβ receptors, BMP receptors, MEKKs,ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub,Myt 1, Wee1, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3,p90Rsks, p70S6Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs,MNKs, AMPKs, MELK, MARKs, Chk1, Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3,IKKs, Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinaseclass 1, class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM,ATR, Receptor protein tyrosine phosphatases (RPTPs), LAR phosphatase,CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinasephosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25phosphatases, Low molecular weight tyrosine phosphatase, Eyes absent(EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serinephosphatases, PP2A, PP2B, PP2C, PP1, PP5, inositol phosphatases, PTEN,SHIPs, myotubularins, phosphoinositide kinases, phopsholipases,prostaglandin synthases, 5-lipoxygenase, sphingosine kinases,sphingomyelinases, adaptor/scaffold proteins, Shc, Grb2, BLNK, LAT, Bcell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL,GAD, Nck, Grb2 associated binder (GAB), Fas associated death domain(FADD), TRADD, TRAF2, RIP, T-Cell leukemia family, IL-2, IL-4, IL-8,IL-6, interferon γ, interferon α, suppressors of cytokine signaling(SOCs), Cbl, SCF ubiquitination ligase complex, APC/C, adhesionmolecules, integrins, Immunoglobulin-like adhesion molecules, selectins,cadherins, catenins, focal adhesion kinase, p130CAS, fodrin, actin,paxillin, myosin, myosin binding proteins, tubulin, eg5/KSP, CENPs,β-adrenergic receptors, muscarinic receptors, adenylyl cyclasereceptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras, Ran,Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam, Sos, Dbl, PRK, TSC1,2,Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Caspase 3, Caspase 6,Caspase 7, Caspase 8, Caspase 9, Bcl-2, Mcl-1, Bcl-XL, Bcl-w, Bcl-B, Al,Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma, IAPs, XIAP,Smac, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D, Cyclin E, Cyclin A,Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecular chaperones, Hsp90s,Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAa Carboxylase, ATP citratelyase, nitric oxide synthase, caveolins, endosomal sorting complexrequired for transport (ESCRT) proteins, vesicular protein sorting(Vsps), hydroxylases, prolyl-hydroxylases PHD-1, 2 and 3, asparaginehydroxylase FIH transferases, Pin1 prolyl isomerase, topoisomerases,deacetylases, Histone deacetylases, sirtuins, histone acetylases,CBP/P300 family, MYST family, ATF2, DNA methyl transferases, HistoneH3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-1, p53, Hdm, PTEN,ubiquitin proteases, urokinase-type plasminogen activator (uPA) and uPAreceptor (uPAR) system, cathepsins, metalloproteinases, esterases,hydrolases, separase, potassium channels, sodium channels, multi-drugresistance proteins, P-Gycoprotein, nucleoside transporters, Ets, Elk,SMADs, Rel-A (p65-NFKB), CREB, NFAT, ATF-2, AFT, Myc, Fos, Sp1, Egr-1,T-bet, β-catenin, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1, β-catenin, FOXOSTAT1, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-1, HMGA, pS6, 4EPB-1,eIF4E-binding protein, RNA polymerase, initiation factors, elongationfactors.

In a further embodiment, the protein is selected from the groupconsisting of Erk, Syk, Zap70, Lyn, Btk, BLNK, Cbl, PLCγ2, Akt, RelA,p38, S6. In another embodiment, the protein is S6.

Binding Element

In some embodiments of the invention, the activation state of anactivatable element is determined by contacting a cell with a bindingelement that is specific for an activation state of the activatableelement. The term “Binding element” includes any molecule, e.g.,peptide, nucleic acid, small organic molecule which is capable ofdetecting an activation state of an activatable element over anotheractivation state of the activatable element.

In some embodiments, the binding element is a peptide, polypeptide,oligopeptide or a protein. The peptide, polypeptide, oligopeptide orprotein may be made up of naturally occurring amino acids and peptidebonds, or synthetic peptidomimetic structures. Thus “amino acid”, or“peptide residue”, as used herein include both naturally occurring andsynthetic amino acids. For example, homo-phenylalanine, citrulline andnoreleucine are considered amino acids for the purposes of theinvention. The side chains may be in either the (R) or the (S)configuration. In some embodiments, the amino acids are in the (S) orL-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradation. Proteins including non-naturally occurringamino acids may be synthesized or in some cases, made recombinantly; seevan Hest et al., FEBS Lett 428:(1-2) 68-70 May 22, 1998 and Tang et al.,Abstr. Pap Am. Chem. S218: U138 Part 2 Aug. 22, 1999, both of which areexpressly incorporated by reference herein.

Methods of the present invention may be used to detect any particularactivatable element in a sample that is antigenically detectable andantigenically distinguishable from other activatable element which ispresent in the sample. For example, as demonstrated (see, e.g., theExamples) and described herein, the activation state-specific antibodiesof the present invention can be used in the present methods to identifydistinct signaling cascades of a subset or subpopulation of complex cellpopulations; and the ordering of protein activation (e.g., kinaseactivation) in potential signaling hierarchies. Hence, in someembodiments the expression and phosphorylation of one or morepolypeptides are detected and quantified using methods of the presentinvention. In some embodiments, the expression and phosphorylation ofone or more polypeptides that are cellular components of a cellularpathway are detected and quantified using methods of the presentinvention. As used herein, the term “activation state-specific antibody”or “activation state antibody” or grammatical equivalents thereof, referto an antibody that specifically binds to a corresponding and specificantigen. Preferably, the corresponding and specific antigen is aspecific form of an activatable element. Also preferably, the binding ofthe activation state-specific antibody is indicative of a specificactivation state of a specific activatable element.

In some embodiments, the binding element is an antibody. In someembodiment, the binding element is an activation state-specificantibody.

The term “antibody” includes full length antibodies and antibodyfragments, and may refer to a natural antibody from any organism, anengineered antibody, or an antibody generated recombinantly forexperimental, therapeutic, or other purposes as further defined below.Examples of antibody fragments, as are known in the art, such as Fab,Fab′, F(ab′)2, Fv, scFv, or other antigen-binding subsequences ofantibodies, either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA technologies. The term“antibody” comprises monoclonal and polyclonal antibodies. Antibodiescan be antagonists, agonists, neutralizing, inhibitory, or stimulatory.

The antibodies of the present invention may be nonhuman, chimeric,humanized, or fully human. For a description of the concepts of chimericand humanized antibodies see Clark et al., 2000 and references citedtherein (Clark, 2000, Immunol Today 21:397-402). Chimeric antibodiescomprise the variable region of a nonhuman antibody, for example VH andVL domains of mouse or rat origin, operably linked to the constantregion of a human antibody (see for example U.S. Pat. No. 4,816,567). Insome embodiments, the antibodies of the present invention are humanized.By “humanized” antibody as used herein is meant an antibody comprising ahuman framework region (FR) and one or more complementarity determiningregions (CDR's) from a non-human (usually mouse or rat) antibody. Thenon-human antibody providing the CDR's is called the “donor” and thehuman immunoglobulin providing the framework is called the “acceptor”.Humanization relies principally on the grafting of donor CDRs ontoacceptor (human) VL and VH frameworks (Winter U.S. Pat. No. 5,225,539).This strategy is referred to as “CDR grafting”. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat.No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 6,180,370; U.S.Pat. No. 5,859,205; U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,054,297;U.S. Pat. No. 6,407,213). The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region,typically that of a human immunoglobulin, and thus will typicallycomprise a human Fc region. Methods for humanizing non-human antibodiesare well known in the art, and can be essentially performed followingthe method of Winter and co-workers (Jones et al., 1986, Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; Verhoeyen etal., 1988, Science, 239:1534-1536). Additional examples of humanizedmurine monoclonal antibodies are also known in the art, for exampleantibodies binding human protein C (O'Connor et al., 1998, Protein Eng11:321-8), interleukin 2 receptor (Queen et al., 1989, Proc Natl AcadSci, USA 86:10029-33), and human epidermal growth factor receptor 2(Carter et al., 1992, Proc Natl. Acad Sci USA 89:4285-9). In analternate embodiment, the antibodies of the present invention may befully human, that is the sequences of the antibodies are completely orsubstantially human. A number of methods are known in the art forgenerating fully human antibodies, including the use of transgenic mice(Bruggemann et al., 1997, Curr Opin Biotechnol 8:455-458) or humanantibody libraries coupled with selection methods (Griffiths et al.,1998, Curr Opin Biotechnol 9:102-108).

Specifically included within the definition of “antibody” areaglycosylated antibodies. By “aglycosylated antibody” as used herein ismeant an antibody that lacks carbohydrate attached at position 297 ofthe Fc region, wherein numbering is according to the EU system as inKabat. The aglycosylated antibody may be a deglycosylated antibody,which is an antibody for which the Fc carbohydrate has been removed, forexample chemically or enzymatically. Alternatively, the aglycosylatedantibody may be a nonglycosylated or unglycosylated antibody, that is anantibody that was expressed without Fc carbohydrate, for example bymutation of one or residues that encode the glycosylation pattern or byexpression in an organism that does not attach carbohydrates toproteins, for example bacteria.

As pointed out above, activation state specific antibodies can be usedto detect kinase activity, however additional means for determiningkinase activation are provided by the present invention. For example,substrates that are specifically recognized by protein kinases andphosphorylated thereby are known. Antibodies that specifically bind tosuch phosphorylated substrates but do not bind to suchnon-phosphorylated substrates (phospho-substrate antibodies) may be usedto determine the presence of activated kinase in a sample.

In a further embodiment, an element activation profile is determinedusing a multiplicity of activation state antibodies that have beenimmobilized. Antibodies may be non-diffusibly bound to an insolublesupport having isolated sample-receiving areas (e.g. a microtiter plate,an array, etc.). The insoluble supports may be made of any compositionto which the compositions can be bound, is readily separated fromsoluble material, and is otherwise compatible with the overall method ofscreening. The surface of such supports may be solid or porous and ofany convenient shape. Examples of suitable insoluble supports includemicrotiter plates, arrays, membranes, and beads. These are typicallymade of glass, plastic (e.g., polystyrene), polysaccharides, nylon ornitrocellulose, Teflon™, etc. Microtiter plates and arrays areespecially convenient because a large number of assays can be carriedout simultaneously, using small amounts of reagents and samples. In somecases magnetic beads and the like are included.

The particular manner of binding of the composition is not crucial solong as it is compatible with the reagents and overall methods of theinvention, maintains the activity of the composition and isnondiffusable. Methods of binding include the use of antibodies (whichdo not sterically block either the ligand binding site or activationsequence when the protein is bound to the support), direct binding to“sticky” or ionic supports, chemical crosslinking, the synthesis of theantibody on the surface, etc. Following binding of the antibody, excessunbound material is removed by washing. The sample receiving areas maythen be blocked through incubation with bovine serum albumin (BSA),casein or other innocuous protein or other moiety.

The antigenicity of an activated isoform of an activatable element isdistinguishable from the antigenicity of non-activated isoform of anactivatable element or from the antigenicity of an isoform of adifferent activation state. In some embodiments, an activated isoform ofan element possesses an epitope that is absent in a non-activatedisoform of an element, or vice versa. In some embodiments, thisdifference is due to covalent addition of moieties to an element, suchas phosphate moieties, or due to a structural change in an element, asthrough protein cleavage, or due to an otherwise induced conformationalchange in an element which causes the element to present the samesequence in an antigenically distinguishable way. In some embodiments,such a conformational change causes an activated isoform of an elementto present at least one epitope that is not present in a non-activatedisoform, or to not present at least one epitope that is presented by anon-activated isoform of the element. In some embodiments, the epitopesfor the distinguishing antibodies are centered around the active site ofthe element, although as is known in the art, conformational changes inone area of an element may cause alterations in different areas of theelement as well.

Many antibodies, many of which are commercially available (for example,see Cell Signaling Technology, www.cellsignal.com, the contents whichare incorporated herein by reference) have been produced whichspecifically bind to the phosphorylated isoform of a protein but do notspecifically bind to a non-phosphorylated isoform of a protein. Manysuch antibodies have been produced for the study of signal transducingproteins which are reversibly phosphorylated. Particularly, many suchantibodies have been produced which specifically bind to phosphorylated,activated isoforms of protein. Examples of proteins that can be analyzedwith the methods described herein include, but are not limited to,kinases, HER receptors, PDGF receptors, Kit receptor, FGF receptors, Ephreceptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor,Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn,Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF,Mos, Lim kinase, ILK, Tpl, ALK, TGFβ receptors, BMP receptors, MEKKs,ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub,Myt 1, Wee1, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3,p90Rsks, p70S6Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs,MNKs, AMPKs, MELK, MARKs, Chk1, Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3,IKKs, Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinaseclass 1, class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM,ATR, phosphatases, Receptor protein tyrosine phosphatases (RPTPs), LARphosphatase, CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs,MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs),CDC25 phosphatases, Low molecular weight tyrosine phosphatase, Eyesabsent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serinephosphatases, PP2A, PP2B, PP2C, PP1, PP5, inositol phosphatases, PTEN,SHIPs, myotubularins, lipid signaling, phosphoinositide kinases,phopsholipases, prostaglandin synthases, 5-lipoxygenase, sphingosinekinases, sphingomyelinases, adaptor/scaffold proteins, Shc, Grb2, BLNK,LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk,CrkL, GAD, Nck, Grb2 associated binder (GAB), Fas associated deathdomain (FADD), TRADD, TRAF2, RIP, T-Cell leukemia family, cytokines,IL-2, IL-4, IL-8, IL-6, interferon γ, interferon α, cytokine regulators,suppressors of cytokine signaling (SOCs), ubiquitination enzymes, Cbl,SCF ubiquitination ligase complex, APC/C, adhesion molecules, integrins,Immunoglobulin-like adhesion molecules, selectins, cadherins, catenins,focal adhesion kinase, p130CAS, cytoskeletal/contractile proteins,fodrin, actin, paxillin, myosin, myosin binding proteins, tubulin,eg5/KSP, CENPs, heterotrimeric G proteins, β-adrenergic receptors,muscarinic receptors, adenylyl cyclase receptors, small molecular weightGTPases, H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB,guanine nucleotide exchange factors, Vav, Tiam, Sos, Dbl, PRK, TSC1,2,GTPase activating proteins, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases,Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9,proteins involved in apoptosis, Bcl-2, Mcl-1, Bcl-XL, Bcl-w, Bcl-B, Al,Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma, IAPs, XIAP,Smac, cell cycle regulators, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D,Cyclin E, Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecularchaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAaCarboxylase, ATP citrate lyase, nitric oxide synthase, vesiculartransport proteins, caveolins, endosomal sorting complex required fortransport (ESCRT) proteins, vesicular protein sorting (Vsps),hydroxylases, prolyl-hydroxylases PHD-1, 2 and 3, asparagine hydroxylaseFIH transferases, isomerases, Pin1 prolyl isomerase, topoisomerases,deacetylases, Histone deacetylases, sirtuins, acetylases, histoneacetylases, CBP/P300 family, MYST family, ATF2, methylases, DNA methyltransferases, demethylases, Histone H3K4 demethylases, H3K27, JHDM2A,UTX, tumor suppressor genes, VHL, WT-1, p53, Hdm, PTEN, proteases,ubiquitin proteases, urokinase-type plasminogen activator (uPA) and uPAreceptor (uPAR) system, cathepsins, metalloproteinases, esterases,hydrolases, separase, ion channels, potassium channels, sodium channels,molecular transporters, multi-drug resistance proteins, P-Gycoprotein,nucleoside transporters, transcription factors/DNA binding proteins,Ets, Elk, SMADs, Rel-A (p65-NFKB), CREB, NFAT, ATF-2, AFT, Myc, Fos,Sp1, Egr-1, T-bet, β-catenin, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1,β-catenin, FOXO STAT1, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-1, HMGA,regulators of translation, pS6, 4EPB-1, eIF4E-binding protein,regulators of transcription, RNA polymerase, initiation factors,elongation factors. In some embodiments, the protein is S6.

In addition to activatable elements, in some embodiments cells areclassified, at least in part, based on cell surface markers. Antibodiesto such markers are well-known and commercially available. Forhematological pre-pathological and pathological conditions the cellsurface markers of interest that may be used in the methods of theinvention include CD2, CD3, CD4, CD5, CD7, CD9, CD10, CD11, CD11b, CD13,CD14, CD15, cCD15, CD19, CD20, CD21, CD22, CD23, CD24, CD31, CD33, CD34,CD36, CD37, CD38, CD39, CD40, CD43, CD44, CD45, cCD45, CD48, CD54, CD56,CD61, CD64, CD65, CD70, CD79b, CD81, CD87, CD116, CD117, CD133, CD135,CD235a, Integrinβ7, CXCR5, LAIR-1, CCR6, kappa light chain, lambda lightchain, HLA-DR, MPO, LF, and TdT, and combinations thereof.

For pre-pathological and pathological solid cancer conditions, the cellsurface markers of interest that may be used in the methods of theinvention include, but are not limited to cell adhesion molecule(EpCAM), also known as epithelial-specific antigen (ESA),carcinoembryonic antigen (CEA), fetal oncogene platelet derived growthfactor receptor (PDGFR), epidermal growth factor receptors (EGFR), Her2,Her3, Her 4, cKit, fibroblast growth factor receptor (FGFR,), insulinlike growth factor 1 receptor (IGF1R,) insulin receptor (IR), vascularendothelial growth factor receptor 1, (VEGFR1), VEGFR2, VEGFR3, TIERs,Ephs, Integrin family, and cadherins.

In some embodiments, an epitope-recognizing fragment of an activationstate antibody rather than the whole antibody is used. In someembodiments, the epitope-recognizing fragment is immobilized. In someembodiments, the antibody light chain that recognizes an epitope isused. A recombinant nucleic acid encoding a light chain gene productthat recognizes an epitope may be used to produce such an antibodyfragment by recombinant means well known in the art.

Non-activation state antibodies may also be used in the presentinvention. In some embodiments, non-activation state antibodies bind toepitopes in both activated and non-activated forms of an element. Suchantibodies may be used to determine the amount of non-activated plusactivated element in a sample. In some embodiments, non-activation stateantibodies bind to epitopes present in non-activated forms of an elementbut absent in activated forms of an element. Such antibodies may be usedto determine the amount of non-activated element in a sample. Both typesof non-activation state antibodies may be used to determine if a changein the amount of activation state element, for example from samplesbefore and after treatment with a candidate bioactive agent as describedherein, coincide with changes in the amount of non-activation stateelement. For example, such antibodies can be used to determine whetheran increase in activated element is due to activation of non-activationstate element, or due to increased expression of the element, or both.

In some embodiments, antibodies are immobilized using beads analogous tothose known and used for standardization in flow cytometry. Attachmentof a multiplicity of activation state specific antibodies to beads maybe done by methods known in the art and/or described herein. Suchconjugated beads may be contacted with sample, preferably cell extract,under conditions that allow for a multiplicity of activated elements, ifpresent, to bind to the multiplicity of immobilized antibodies. A secondmultiplicity of antibodies comprising non-activation state antibodieswhich are uniquely labeled may be added to the immobilized activationstate specific antibody-activated element complex and the beads may besorted by FACS on the basis of the presence of each label, wherein thepresence of label indicates binding of corresponding second antibody andthe presence of corresponding activated element.

In alternative embodiments of the instant invention, aromatic aminoacids of protein binding elements may be replaced with D- orL-naphylalanine, D- or L-phenylglycine, D- or L-2-thieneylalanine, D- orL-1-, 2-, 3- or 4-pyreneylalanine, D- or L-3-thieneylalanine, D- orL-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- orL-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine,D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine,D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- orL-p-methoxybiphenylphenylalanine, D- or L-2-indole(alkyl)alanines, andD- or L-alkylalanines where alkyl may be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, and non-acidic amino acids of C1-C20.

Acidic amino acids can be substituted with non-carboxylate amino acidswhile maintaining a negative charge, and derivatives or analogs thereof,such as the non-limiting examples of (phosphono)alanine, glycine,leucine, isoleucine, threonine, or serine; or sulfated (e.g., —SO3H)threonine, serine, or tyrosine.

Other substitutions may include nonnatural hydroxylated amino acids maymade by combining “alkyl” with any natural amino acid. The term “alkyl”as used herein refers to a branched or unbranched saturated hydrocarbongroup of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl,isoptopyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl,hexadecyl, eicosyl, tetracisyl and the like. Alkyl includes heteroalkyl,with atoms of nitrogen, oxygen and sulfur. In some embodiments, alkylgroups herein contain 1 to 12 carbon atoms. Basic amino acids may besubstituted with alkyl groups at any position of the naturally occurringamino acids lysine, arginine, ornithine, citrulline, or(guanidino)-acetic acid, or other (guanidino)alkyl-acetic acids, where“alkyl” is define as above. Nitrile derivatives (e.g., containing theCN-moiety in place of COOH) may also be substituted for asparagine orglutamine, and methionine sulfoxide may be substituted for methionine.Methods of preparation of such peptide derivatives are well known to oneskilled in the art.

In addition, any amide linkage in any of the polypeptides may bereplaced by a ketomethylene moiety. Such derivatives are expected tohave the property of increased stability to degradation by enzymes, andtherefore possess advantages for the formulation of compounds which mayhave increased in vivo half lives, as administered by oral, intravenous,intramuscular, intraperitoneal, topical, rectal, intraocular, or otherroutes.

Additional amino acid modifications of amino acids of variantpolypeptides of to the present invention may include the following:Cysteinyl residues may be reacted with alpha-haloacetates (andcorresponding amines), such as 2-chloroacetic acid or chloroacetamide,to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinylresidues may also be derivatized by reaction with compounds such asbromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues may be derivatized by reaction with compounds such asdiethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain, and para-bromophenacyl bromide mayalso be used; e.g., where the reaction is preferably performed in 0.1Msodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds suchas succinic or other carboxylic acid anhydrides. Derivatization withthese agents is expected to have the effect of reversing the charge ofthe lysinyl residues.

Other suitable reagents for derivatizing alpha-amino-containing residuesinclude compounds such as imidoesters, e.g., as methyl picolinimidate;pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate. Arginyl residues may bemodified by reaction with one or several conventional reagents, amongthem phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrinaccording to known method steps. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group. The specific modification of tyrosyl residues perse is well known, such as for introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane.

N-acetylimidizol and tetranitromethane may be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Carboxyl sidegroups (aspartyl or glutamyl) may be selectively modified by reactionwith carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholiny-1-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermoreaspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of the present invention.

In some embodiments, the activation state-specific binding element is apeptide comprising a recognition structure that binds to a targetstructure on an activatable protein. A variety of recognition structuresare well known in the art and can be made using methods known in theart, including by phage display libraries (see e.g., Gururaja et al.Chem. Biol. (2000) 7:515-27; Houimel et al., Eur. J. Immunol. (2001)31:3535-45; Cochran et al. J. Am. Chem. Soc. (2001) 123:625-32; Houimelet al. Int. J. Cancer (2001) 92:748-55, each incorporated herein byreference). Further, fluorophores can be attached to such antibodies foruse in the methods of the present invention.

A variety of recognitions structures are known in the art (e.g., Cochranet al., J. Am. Chem. Soc. (2001) 123:625-32; Boer et al., Blood (2002)100:467-73, each expressly incorporated herein by reference)) and can beproduced using methods known in the art (see e.g., Boer et al., Blood(2002) 100:467-73; Gualillo et al., Mol. Cell Endocrinol. (2002)190:83-9, each expressly incorporated herein by reference)), includingfor example combinatorial chemistry methods for producing recognitionstructures such as polymers with affinity for a target structure on anactivatable protein (see e.g., Barn et al., J. Comb. Chem. (2001)3:534-41; Ju et al., Biotechnol. (1999) 64:232-9, each expresslyincorporated herein by reference). In another embodiment, the activationstate-specific antibody is a protein that only binds to an isoform of aspecific activatable protein that is phosphorylated and does not bind tothe isoform of this activatable protein when it is not phosphorylated ornonphosphorylated. In another embodiment the activation state-specificantibody is a protein that only binds to an isoform of an activatableprotein that is intracellular and not extracellular, or vice versa. In asome embodiment, the recognition structure is an anti-lamininsingle-chain antibody fragment (scFv) (see e.g., Sanz et al., GeneTherapy (2002) 9:1049-53; Tse et al., J. Mol. Biol. (2002) 317:85-94,each expressly incorporated herein by reference).

In some embodiments the binding element is a nucleic acid. The term“nucleic acid” include nucleic acid analogs, for example, phosphoramide(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J.Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487(1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am.Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al.,Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);Carlsson et al., Nature 380:207 (1996), all of which are incorporated byreference). Other analog nucleic acids include those with positivebackbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995);non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240,5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed.English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994);Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of additional moieties such as labels, or to increase thestability and half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made. In someembodiments, peptide nucleic acids (PNA) which includes peptide nucleicacid analogs are used. These backbones are substantially non-ionic underneutral conditions, in contrast to the highly charged phosphodiesterbackbone of naturally occurring nucleic acids.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc.

In some embodiments, the binding element is a synthetic compound. Anynumbers of techniques are available for the random and directedsynthesis of a wide variety of organic compounds and biomolecules,including expression of randomized oligonucleotides. See for example WO94/24314, hereby expressly incorporated by reference, which discussesmethods for generating new compounds, including random chemistry methodsas well as enzymatic methods.

Alternatively, some embodiments utilize natural compounds, as bindingelements, in the form of bacterial, fungal, plant and animal extractsthat are available or readily produced.

Additionally, natural or synthetically produced compounds are readilymodified through conventional chemical, physical and biochemical means.Known pharmacological agents may be subjected to directed or randomchemical modifications, including enzymatic modifications, to producebinding elements that may be used in the instant invention.

In some embodiment the binding element is a small organic compound.Binding elements can be synthesized from a series of substrates that canbe chemically modified. “Chemically modified” herein includestraditional chemical reactions as well as enzymatic reactions. Thesesubstrates generally include, but are not limited to, alkyl groups(including alkanes, alkenes, alkynes and heteroalkyl), aryl groups(including arenes and heteroaryl), alcohols, ethers, amines, aldehydes,ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds(including purines, pyrimidines, benzodiazepins, beta-lactams,tetracylines, cephalosporins, and carbohydrates), steroids (includingestrogens, androgens, cortisone, ecodysone, etc.), alkaloids (includingergots, vinca, curare, pyrollizdine, and mitomycines), organometalliccompounds, hetero-atom bearing compounds, amino acids, and nucleosides.Chemical (including enzymatic) reactions may be done on the moieties toform new substrates or binding elements that can then be used in thepresent invention.

In some embodiments the binding element is a carbohydrate. As usedherein the term carbohydrate is meant to include any compound with thegeneral formula (CH₂O)_(n). Examples of carbohydrates are di-, tri- andoligosaccharides, as well polysaccharides such as glycogen, cellulose,and starches.

In some embodiments the binding element is a lipid. As used herein theterm lipid herein is meant to include any water insoluble organicmolecule that is soluble in nonpolar organic solvents. Examples oflipids are steroids, such as cholesterol, and phospholipids such assphingomeylin.

Examples of activatable elements, activation states and methods ofdetermining the activation state of activatable elements are describedin US publication number 20060073474 entitled “Methods and compositionsfor detecting the activation state of multiple proteins in single cells”and US publication number 20050112700 entitled “Methods and compositionsfor risk stratification” the content of which are incorporate here byreference.

These and other elements are known to those of skill in the art. SeeU.S. patent application Ser. Nos. 10/193,462; 10/898,734; 10/346,620;and 11/338,957, all of which are incorporated herein by reference intheir entirety.

Labels

The methods and compositions of the instant invention provide bindingelements comprising a label or tag. By label is meant a molecule thatcan be directly (i.e., a primary label) or indirectly (i.e., a secondarylabel) detected; for example a label can be visualized and/or measuredor otherwise identified so that its presence or absence can be known. Acompound can be directly or indirectly conjugated to a label whichprovides a detectable signal, e.g. radioisotopes, fluorescers, enzymes,antibodies, particles such as magnetic particles, chemiluminescers, orspecific binding molecules, etc. Specific binding molecules includepairs, such as biotin and streptavidin, digoxin and antidigoxin etc.Examples of labels include, but are not limited to, optical fluorescentand chromogenic dyes including labels, label enzymes and radioisotopes.

In some embodiments, one or more binding elements are uniquely label.Using the example of two activation state specific antibodies, by“uniquely labeled” is meant that a first activation state antibodyrecognizing a first activated element comprises a first label, andsecond activation state antibody recognizing a second activated elementcomprises a second label, wherein the first and second labels aredetectable and distinguishable, making the first antibody and the secondantibody uniquely labeled.

In general, labels fall into four classes: a) isotopic labels, which maybe radioactive or heavy isotopes; b) magnetic, electrical, thermallabels; c) colored, optical labels including luminescent, phosphorousand fluorescent dyes or moieties; and d) binding partners. Labels canalso include enzymes (horseradish peroxidase, etc.) and magneticparticles. In some embodiments, the detection label is a primary label.A primary label is one that can be directly detected, such as afluorophore.

Labels include optical labels such as fluorescent dyes or moieties.Fluorophores can be either “small molecule” fluors, or proteinaceousfluors (e.g. green fluorescent proteins and all variants thereof).

Suitable fluorescent labels include, but are not limited to,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640,Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes aredescribed in the 1996 Molecular Probes Handbook by Richard P. Haugland,hereby expressly incorporated by reference. Suitable fluorescent labelsalso include, but are not limited to, green fluorescent protein (GFP;Chalfie, et al., Science 263(5148):802-805 (Feb. 11, 1994); and EGFP;Clontech—Genbank Accession Number U55762), blue fluorescent protein(BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West,8th Floor, Montreal (Quebec) Canada H3H1J9; 2. Stauber, R. H.Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr.Biol. 6:178-182 (1996)), enhanced yellow fluorescent protein (EYFP; 1.Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif.94303), luciferase (Ichiki, et al., J. Immunol. 150(12):5408-5417(1993)), .beta.-galactosidase (Nolan, et al., Proc Natl Acad Sci USA85(8):2603-2607 (April 1988)) and Renilla WO 92/15673; WO 95/07463; WO98/14605; WO 98/26277; WO 99/49019; U.S. Pat. No. 5,292,658; U.S. Pat.No. 5,418,155; U.S. Pat. No. 5,683,888; U.S. Pat. No. 5,741,668; U.S.Pat. No. 5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304;U.S. Pat. No. 5,876,995; and U.S. Pat. No. 5,925,558). All of theabove-cited references are expressly incorporated herein by reference.

In some embodiments, labels for use in the present invention include:Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488,Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633,Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow andR-phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg.), FITC,Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7(Amersham Life Science, Pittsburgh, Pa.). Tandem conjugate protocols forCy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, Cy7APC can be found athttp://www.drmr.com/index.html. Antibodies and labels are commerciallyavailable at Becton Dickinson,http://www.bdbiosciences.com/features/products/display_product.php?keyID=94.Quantitation of fluorescent probe conjugation may be assessed todetermine degree of labeling and protocols including dye spectralproperties are also well known in the art.

In some embodiments, the fluorescent label is a GFP and, morepreferably, a Renilla, Ptilosarcus, or Aequorea species of GFP.

In some embodiments, a secondary detectable label is used. A secondarylabel is one that is indirectly detected; for example, a secondary labelcan bind or react with a primary label for detection, can act on anadditional product to generate a primary label (e.g. enzymes), etc.Secondary labels include, but are not limited to, one of a bindingpartner pair; chemically modifiable moieties; nuclease inhibitors,enzymes such as horseradish peroxidase, alkaline phosphatases,luciferases, etc.

In some embodiments, the secondary label is a binding partner pair. Forexample, the label may be a hapten or antigen, which will bind itsbinding partner. For example, suitable binding partner pairs include,but are not limited to: antigens (such as proteins (including peptides)and small molecules) and antibodies (including fragments thereof (FAbs,etc.)); proteins and small molecules, including biotin/streptavidin;enzymes and substrates or inhibitors; other protein-protein interactingpairs; receptor-ligands; and carbohydrates and their binding partners.Nucleic acid—nucleic acid binding proteins pairs are also useful.Binding partner pairs include, but are not limited to, biotin (orimino-biotin) and streptavidin, digeoxinin and Abs, and Prolinx™reagents.

In some embodiments, the binding partner pair comprises an antigen andan antibody that will specifically bind to the antigen. By “specificallybind” herein is meant that the partners bind with specificity sufficientto differentiate between the pair and other components or contaminantsof the system. The binding should be sufficient to remain bound underthe conditions of the assay, including wash steps to remove non-specificbinding. In some embodiments, the dissociation constants of the pairwill be less than about 10⁻⁴ to 10⁻⁹ M⁻¹, with less than about 10⁻⁵ to10⁻⁹ M⁻¹ being preferred and less than about 10⁻⁷ to 10⁻⁹ M⁻¹ beingparticularly preferred.

In some embodiment, the secondary label is a chemically modifiablemoiety. In this embodiment, labels comprising reactive functional groupsare incorporated into the molecule to be labeled. The functional groupcan then be subsequently labeled (e.g. either before or after the assay)with a primary label. Suitable functional groups include, but are notlimited to, amino groups, carboxy groups, maleimide groups, oxo groupsand thiol groups, with amino groups and thiol groups being particularlypreferred. For example, primary labels containing amino groups can beattached to secondary labels comprising amino groups, for example usinglinkers as are known in the art; for example, homo- orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference).

In some embodiments, multiple fluorescent labels are employed in themethods and compositions of the present invention. In some embodiments,each label is distinct and distinguishable from other labels.

As will be appreciated in the art antibody-label conjugation may beperformed using standard procedures or by usingprotein-protein/protein-dye crosslinking kits from Molecular Probes(Eugene, Oreg.).

In some embodiments, labeled antibodies are used for functional analysisof activatable proteins in cells. In performing such analysis severalareas of the experiment are considered: (1) identification of the propercombination of antibody cocktails for the stains (2), identification ofthe sequential procedure for the staining using the antigens (i.e., theactivatable protein) and antibody clones of interest, and (3) thoroughevaluation of cell culture conditions' effect on cell stimulation.Antigen clone selection is of particular importance for surface antigensof human cells, as different antibody clones yield different result anddo not stain similarly in different protocols. Selection of cell typesand optimization of culture conditions is also a critical component indetecting differences. For example, some cell lines have the ability toadapt to culture conditions and can yield heterogeneous responses.

Alternatively, detection systems based on FRET, discussed in detailbelow, may be used. FRET finds use in the instant invention, forexample, in detecting activation states that involve clustering ormultimerization wherein the proximity of two FRET labels is altered dueto activation. In some embodiments, at least two fluorescent labels areused which are members of a fluorescence resonance energy transfer(FRET) pair.

FRET is phenomenon known in the art wherein excitation of onefluorescent dye is transferred to another without emission of a photon.A FRET pair consists of a donor fluorophore and an acceptor fluorophore.The fluorescence emission spectrum of the donor and the fluorescenceabsorption spectrum of the acceptor must overlap, and the two moleculesmust be in close proximity. The distance between donor and acceptor atwhich 50% of donors are deactivated (transfer energy to the acceptor) isdefined by the Forster radius (Ro), which is typically 10-100 Å. Changesin the fluorescence emission spectrum comprising FRET pairs can bedetected, indicating changes in the number of that are in closeproximity (i.e., within 100 521 of each other). This will typicallyresult from the binding or dissociation of two molecules, one of whichis labeled with a FRET donor and the other of which is labeled with aFRET acceptor, wherein such binding brings the FRET pair in closeproximity. Binding of such molecules will result in an increasedfluorescence emission of the acceptor and/or quenching of thefluorescence emission of the donor.

FRET pairs (donor/acceptor) useful in the invention include, but are notlimited to, EDANS/fluorescein, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein/Cy5, fluorescein/Cy 5.5 and fluorescein/LC Red 705.

In some embodiments when FRET is used, a fluorescent donor molecule anda nonfluorescent acceptor molecule (“quencher”) may be employed. In thisapplication, fluorescent emission of the donor will increase whenquencher is displaced from close proximity to the donor and fluorescentemission will decrease when the quencher is brought into close proximityto the donor. Useful quenchers include, but are not limited to, TAMRA,DABCYL, QSY 7 and QSY 33. Useful fluorescent donor/quencher pairsinclude, but are not limited to EDANS/DABCYL, Texas Red/DABCYL,BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL andfluorescein/QSY 7 dye.

The skilled artisan will appreciate that FRET and fluorescence quenchingallow for monitoring of binding of labeled molecules over time,providing continuous information regarding the time course of bindingreactions.

Preferably, changes in the degree of FRET are determined as a functionof the change in the ratio of the amount of fluorescence from the donorand acceptor moieties, a process referred to as “ratioing.” Changes inthe absolute amount of substrate, excitation intensity, and turbidity orother background absorbances in the sample at the excitation wavelengthaffect the intensities of fluorescence from both the donor and acceptorapproximately in parallel. Therefore the ratio of the two emissionintensities is a more robust and preferred measure of cleavage thaneither intensity alone.

The ratio-metric fluorescent reporter system described herein hassignificant advantages over existing reporters for protein integrationanalysis, as it allows sensitive detection and isolation of bothexpressing and non-expressing single living cells. In some embodiments,the assay system uses a non-toxic, non-polar fluorescent substrate thatis easily loaded and then trapped intracellularly. Modification of thefluorescent substrate by a cognate protein yields a fluorescent emissionshift as substrate is converted to product. Because the reporter readoutis ratiometric it is unique among reporter protein assays in that itcontrols for variables such as the amount of substrate loaded intoindividual cells. The stable, easily detected, intracellular readouteliminates the need for establishing clonal cell lines prior toexpression analysis. This system and other analogous flow sortingsystems can be used to isolate cells having a particular receptorelement clustering and/or activation profile from pools of millions ofviable cells.

The methods and composition of the present invention may also make useof label enzymes. By label enzyme is meant an enzyme that may be reactedin the presence of a label enzyme substrate that produces a detectableproduct. Suitable label enzymes for use in the present invention includebut are not limited to, horseradish peroxidase, alkaline phosphatase andglucose oxidase. Methods for the use of such substrates are well knownin the art. The presence of the label enzyme is generally revealedthrough the enzyme's catalysis of a reaction with a label enzymesubstrate, producing an identifiable product. Such products may beopaque, such as the reaction of horseradish peroxidase with tetramethylbenzedine, and may have a variety of colors. Other label enzymesubstrates, such as Luminol (available from Pierce Chemical Co.), havebeen developed that produce fluorescent reaction products. Methods foridentifying label enzymes with label enzyme substrates are well known inthe art and many commercial kits are available. Examples and methods forthe use of various label enzymes are described in Savage et al.,Previews 247:6-9 (1998), Young, J. Virol. Methods 24:227-236 (1989),which are each hereby incorporated by reference in their entirety.

By radioisotope is meant any radioactive molecule. Suitableradioisotopes for use in the invention include, but are not limited to¹⁴C, ³H, ³²P, ³³p, ³⁵S, ¹²⁵I, and ¹³¹I. The use of radioisotopes aslabels is well known in the art.

As mentioned, labels may be indirectly detected, that is, the tag is apartner of a binding pair. By “partner of a binding pair” is meant oneof a first and a second moiety, wherein the first and the second moietyhave a specific binding affinity for each other. Suitable binding pairsfor use in the invention include, but are not limited to,antigens/antibodies (for example, digoxigenin/anti-digoxigenin,dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl,Fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, andrhodamine anti-rhodamine), biotin/avidin (or biotin/streptavidin) andcalmodulin binding protein (CBP)/calmodulin. Other suitable bindingpairs include polypeptides such as the FLAG-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255: 192-194 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)] and the antibodies each thereto. As will beappreciated by those in the art, binding pair partners may be used inapplications other than for labeling, as is described herein.

As will be appreciated by those in the art, a partner of one bindingpair may also be a partner of another binding pair. For example, anantigen (first moiety) may bind to a first antibody (second moiety) thatmay, in turn, be an antigen for a second antibody (third moiety). Itwill be further appreciated that such a circumstance allows indirectbinding of a first moiety and a third moiety via an intermediary secondmoiety that is a binding pair partner to each.

As will be appreciated by those in the art, a partner of a binding pairmay comprise a label, as described above. It will further be appreciatedthat this allows for a tag to be indirectly labeled upon the binding ofa binding partner comprising a label. Attaching a label to a tag that isa partner of a binding pair, as just described, is referred to herein as“indirect labeling”.

By “surface substrate binding molecule” or “attachment tag” andgrammatical equivalents thereof is meant a molecule have bindingaffinity for a specific surface substrate, which substrate is generallya member of a binding pair applied, incorporated or otherwise attachedto a surface. Suitable surface substrate binding molecules and theirsurface substrates include, but are not limited to poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags and Nickelsubstrate; the Glutathione-S Transferase tag and its antibody substrate(available from Pierce Chemical); the flu HA tag polypeptide and itsantibody 12CA5 substrate [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodysubstrates thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody substrate [Paborsky et al., Protein Engineering,3(6):547-553 (1990)]. In general, surface binding substrate moleculesuseful in the present invention include, but are not limited to,polyhistidine structures (His-tags) that bind nickel substrates,antigens that bind to surface substrates comprising antibody, haptensthat bind to avidin substrate (e.g., biotin) and CBP that binds tosurface substrate comprising calmodulin.

Production of antibody-embedded substrates is well known; see Slinkin etal., Bioconj. Chem., 2:342-348 (1991); Torchilin et al., supra;Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et al., CancerRes. 54:6176-6185 (1994); and Wilbur et al., Bioconjugate Chem.5:220-235 (1994) (all of which are hereby expressly incorporated byreference), and attachment of or production of proteins with antigens isdescribed above. Calmodulin-embedded substrates are commerciallyavailable, and production of proteins with CBP is described in Simcox etal., Strategies 8:40-43 (1995), which is hereby incorporated byreference in its entirety.

As will be appreciated by those in the art, tag-components of theinvention can be made in various ways, depending largely upon the formof the tag. Components of the invention and tags are preferably attachedby a covalent bond.

The production of tag-polypeptides by recombinant means when the tag isalso a polypeptide is described below. Production of tag-labeledproteins is well known in the art and kits for such production arecommercially available (for example, from Kodak and Sigma). Examples oftag labeled proteins include, but are not limited to, a Flag-polypeptideand His-polypeptide. Methods for the production and use of tag-labeledproteins are found, for example, in Winston et al., Genes and Devel.13:270-283 (1999), incorporated herein in its entirety, as well asproduct handbooks provided with the above-mentioned kits.

Biotinylation of target molecules and substrates is well known, forexample, a large number of biotinylation agents are known, includingamine-reactive and thiol-reactive agents, for the biotinylation ofproteins, nucleic acids, carbohydrates, carboxylic acids; see chapter 4,Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby incorporated byreference. A biotinylated substrate can be attached to a biotinylatedcomponent via avidin or streptavidin. Similarly, a large number ofhaptenylation reagents are also known (Id.).

Methods for labeling of proteins with radioisotopes are known in theart. For example, such methods are found in Ohta et al., Molec. Cell3:535-541 (1999), which is hereby incorporated by reference in itsentirety.

Production of proteins having tags by recombinant means is well known,and kits for producing such proteins are commercially available. Forexample, such a kit and its use are described in the QIAexpress Handbookfrom Qiagen by Joanne Crowe et al., hereby expressly incorporated byreference.

The functionalization of labels with chemically reactive groups such asthiols, amines, carboxyls, etc. is generally known in the art. In someembodiments, the tag is functionalized to facilitate covalentattachment. The covalent attachment of the tag may be either direct orvia a linker. In one embodiment, the linker is a relatively shortcoupling moiety, which is used to attach the molecules. A couplingmoiety may be synthesized directly onto a component of the invention andcontains at least one functional group to facilitate attachment of thetag. Alternatively, the coupling moiety may have at least two functionalgroups, which are used to attach a functionalized component to afunctionalized tag, for example. In an additional embodiment, the linkeris a polymer. In this embodiment, covalent attachment is accomplishedeither directly, or through the use of coupling moieties from thecomponent or tag to the polymer. In some embodiments, the covalentattachment is direct, that is, no linker is used. In this embodiment,the component preferably contains a functional group such as acarboxylic acid that is used for direct attachment to the functionalizedtag. It should be understood that the component and tag may be attachedin a variety of ways, including those listed above. In some embodiments,the tag is attached to the amino or carboxl terminus of the polypeptide.As will be appreciated by those in the art, the above description of thecovalent attachment of a label applies to the attachment of virtuallyany two molecules of the present disclosure.

In some embodiments, the tag is functionalized to facilitate covalentattachment, as is generally outlined above. Thus, a wide variety of tagsare commercially available which contain functional groups, including,but not limited to, isothiocyanate groups, amino groups, haloacetylgroups, maleimides, succinimidyl esters, and sulfonyl halides, all ofwhich may be used to covalently attach the tag to a second molecule, asis described herein. The choice of the functional group of the tag willdepend on the site of attachment to either a linker, as outlined aboveor a component of the invention. Thus, for example, for direct linkageto a carboxylic acid group of a protein, amino modified or hydrazinemodified tags will be used for coupling via carbodiimide chemistry, forexample using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimi-de (EDAC) asis known in the art (see Set 9 and Set 11 of the Molecular ProbesCatalog, supra; see also the Pierce 1994 Catalog and Handbook, pagesT-155 to T-200, both of which are hereby incorporated by reference). Inone embodiment, the carbodiimide is first attached to the tag, such asis commercially available for many of the tags described herein.

Detection

In practicing the methods of this invention, the detection of the statusof the one or more activatable elements can be carried out by a person,such as a technician in the laboratory. Alternatively, the detection ofthe status of the one or more activatable elements can be carried outusing automated systems. In either case, the detection of the status ofthe one or more activatable elements for use according to the methods ofthis invention can be performed according to standard techniques andprotocols well-established in the art.

One or more activatable elements can be detected and/or quantified byany method that detect and/or quantitates the presence of theactivatable element of interest. Such methods may includeradioimmunoassay (RIA) or enzyme linked immunoabsorbance assay (ELISA),immunohistochemistry, immunofluorescent histochemistry with or withoutconfocal microscopy, reversed phase assays, homogeneous enzymeimmunoassays, and related non-enzymatic techniques, Western blots, wholecell staining, immunoelectronmicroscopy, nucleic acid amplification,gene array, protein array, mass spectrometry, patch clamp, 2-dimensionalgel electrophoresis, differential display gel electrophoresis,microsphere-based multiplex protein assays, label-free cellular assaysand flow cytometry, etc. U.S. Pat. No. 4,568,649 describes liganddetection systems, which employ scintillation counting. These techniquesare particularly useful for modified protein parameters. Cell readoutsfor proteins and other cell determinants can be obtained usingfluorescent or otherwise tagged reporter molecules. Flow cytometrymethods are useful for measuring intracellular parameters.

In some embodiments, the present invention provides methods fordetermining an activatable element's activation profile for a singlecell. The methods may comprise analyzing cells by flow cytometry on thebasis of the activation state of at least two activatable elements.Binding elements (e.g. activation state-specific antibodies) are used toanalyze cells on the basis of activatable element activation state, andcan be detected as described below. Alternatively, non-binding elementssystems as described above can be used in any system described herein.

When using fluorescent labeled components in the methods andcompositions of the present invention, it will recognized that differenttypes of fluorescent monitoring systems, e.g., FACS systems, can be usedto practice the invention. In some embodiments, FACS systems are used orsystems dedicated to high throughput screening, e.g. 96 well or greatermicrotiter plates. Methods of performing assays on fluorescent materialsare well known in the art and are described in, e.g., Lakowicz, J. R.,Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983);Herman, B., Resonance energy transfer microscopy, in: FluorescenceMicroscopy of Living Cells in Culture, Part B, Methods in Cell Biology,vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press(1989), pp. 219-243; Turro, N. J., Modern Molecular Photochemistry,Menlo Park: Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.

Fluorescence in a sample can be measured using a fluorimeter. Ingeneral, excitation radiation, from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescent proteins in the sample emit radiation that has a wavelengththat is different from the excitation wavelength. Collection optics thencollect the emission from the sample. The device can include atemperature controller to maintain the sample at a specific temperaturewhile it is being scanned. According to one embodiment, a multi-axistranslation stage moves a microtiter plate holding a plurality ofsamples in order to position different wells to be exposed. Themulti-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation. In general, known robotic systems andcomponents can be used.

Activation state-specific antibodies can also be labeled with quantumdots as disclosed by Chattopadhyay, P. K. et al. Quantum dotsemiconductor nanocrystals for immunophenotyping by polychromatic flowcytometry. Nat. Med. 12, 972-977 (2006). Quantum dot labels arecommercially available through Invitrogen,http://probes.invitrogen.com/products/qdot/.

Quantum dot labeled antibodies can be used alone or they can be employedin conjunction with organic fluorochrome conjugated antibodies toincrease the total number of labels available. As the number of labeledantibodies increase so does the ability for subtyping known cellpopulations. Additionally, activation state-specific antibodies can belabeled using chelated or caged lanthanides as disclosed by Erkki, J. etal. Lanthanide chelates as new fluorochrome labels for cytochemistry. J.Histochemistry Cytochemistry, 36:1449-1451, 1988, and U.S. Pat. No.7,018,850, entitled Salicylamide-Lanthanide Complexes for Use asLuminescent Markers. Other methods of detecting fluorescence may also beused, e.g., Quantum dot methods (see, e.g., Goldman et al., J. Am. Chem.Soc. (2002) 124:6378-82; Pathak et al. J. Am. Chem. Soc. (2001)123:4103-4; and Remade et al., Proc. Natl. Sci. USA (2000) 18:553-8,each expressly incorporated herein by reference) as well as confocalmicroscopy.

In general, flow cytometry involves the passage of individual cellsthrough the path of a laser beam. The scattering the beam and excitationof any fluorescent molecules attached to, or found within, the cell isdetected by photomultiplier tubes to create a readable output, e.g.size, granularity, or fluorescent intensity.

The detecting, sorting, or isolating step of the methods of the presentinvention can entail fluorescence-activated cell sorting (FACS)techniques, where FACS is used to select cells from the populationcontaining a particular surface marker, or the selection step can entailthe use of magnetically responsive particles as retrievable supports fortarget cell capture and/or background removal. A variety of FACS systemsare known in the art and can be used in the methods of the invention(see e.g., WO99/54494, filed Apr. 16, 1999; U.S. Ser. No. 20010006787,filed Jul. 5, 2001, each expressly incorporated herein by reference).

In some embodiments, a FACS cell sorter (e.g. a FACSVantage™ CellSorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif.) isused to sort and collect cells based on their activation profile(positive cells) in the presence or absence of an increase in activationstate in an activatable element in response to a modulator.

In some embodiments, the cells are first contacted withfluorescent-labeled activation state-specific binding elements (e.g.antibodies) directed against specific activation state of specificactivatable elements. In such an embodiment, the amount of bound bindingelement on each cell can be measured by passing droplets containing thecells through the cell sorter. By imparting an electromagnetic charge todroplets containing the positive cells, the cells can be separated fromother cells. The positively selected cells can then be harvested insterile collection vessels. These cell-sorting procedures are describedin detail, for example, in the FACSVantage™. Training Manual, withparticular reference to sections 3-11 to 3-28 and 10-1 to 10-17, whichis hereby incorporated by reference in its entirety.

In another embodiment, positive cells can be sorted using magneticseparation of cells based on the presence of an isoform of anactivatable element. In such separation techniques, cells to bepositively selected are first contacted with specific binding element(e.g., an antibody or reagent that binds an isoform of an activatableelement). The cells are then contacted with retrievable particles (e.g.,magnetically responsive particles) that are coupled with a reagent thatbinds the specific element. The cell-binding element-particle complexcan then be physically separated from non-positive or non-labeled cells,for example, using a magnetic field. When using magnetically responsiveparticles, the positive or labeled cells can be retained in a containerusing a magnetic filed while the negative cells are removed. These andsimilar separation procedures are described, for example, in the BaxterImmunotherapy Isolex training manual which is hereby incorporated in itsentirety.

In some embodiments, methods for the determination of a receptor elementactivation state profile for a single cell are provided. The methodscomprise providing a population of cells and analyze the population ofcells by flow cytometry. Preferably, cells are analyzed on the basis ofthe activation state of at least two activatable elements. In someembodiments, a multiplicity of activatable element activation-stateantibodies is used to simultaneously determine the activation state of amultiplicity of elements.

In some embodiment, cell analysis by flow cytometry on the basis of theactivation state of at least two elements is combined with adetermination of other flow cytometry readable outputs, such as thepresence of surface markers, granularity and cell size to provide acorrelation between the activation state of a multiplicity of elementsand other cell qualities measurable by flow cytometry for single cells.

As will be appreciated, the present invention also provides for theordering of element clustering events in signal transduction.Particularly, the present invention allows the artisan to construct anelement clustering and activation hierarchy based on the correlation oflevels of clustering and activation of a multiplicity of elements withinsingle cells. Ordering can be accomplished by comparing the activationstate of a cell or cell population with a control at a single timepoint, or by comparing cells at multiple time points to observesubpopulations arising out of the others.

The present invention provides a valuable method of determining thepresence of cellular subsets within cellular populations. Ideally,signal transduction pathways are evaluated in homogeneous cellpopulations to ensure that variances in signaling between cells do notqualitatively nor quantitatively mask signal transduction events andalterations therein. As the ultimate homogeneous system is the singlecell, the present invention allows the individual evaluation of cells toallow true differences to be identified in a significant way.

Thus, the invention provides methods of distinguishing cellular subsetswithin a larger cellular population. As outlined herein, these cellularsubsets often exhibit altered biological characteristics (e.g.activation states, altered response to modulators) as compared to othersubsets within the population. For example, as outlined herein, themethods of the invention allow the identification of subsets of cellsfrom a population such as primary cell populations, e.g. peripheralblood mononuclear cells that exhibit altered responses (e.g. responseassociated with presence of a condition) as compared to other subsets.In addition, this type of evaluation distinguishes between differentactivation states, altered responses to modulators, cell lineages, celldifferentiation states, etc.

As will be appreciated, these methods provide for the identification ofdistinct signaling cascades for both artificial and stimulatoryconditions in complex cell populations, such a peripheral bloodmononuclear cells, or naive and memory lymphocytes.

When necessary, cells are dispersed into a single cell suspension, e.g.by enzymatic digestion with a suitable protease, e.g. collagenase,dispase, etc; and the like. An appropriate solution is used fordispersion or suspension. Such solution will generally be a balancedsalt solution, e.g. normal saline, PBS, Hanks balanced salt solution,etc., conveniently supplemented with fetal calf serum or other naturallyoccurring factors, in conjunction with an acceptable buffer at lowconcentration, generally from 5-25 mM. Convenient buffers include HEPES1phosphate buffers, lactate buffers, etc. The cells may be fixed, e.g.with 3% paraformaldehyde, and are usually permeabilized, e.g. with icecold methanol; HEPES-buffered PBS containing 0.1% saponin, 3% BSA;covering for 2 min in acetone at −200° C.; and the like as known in theart and according to the methods described herein.

In some embodiments, one or more cells are contained in a well of a 96well plate or other commercially available multiwell plate. In analternate embodiment, the reaction mixture or cells are in a FACSmachine. Other multiwell plates useful in the present invention include,but are not limited to 384 well plates and 1536 well plates. Still othervessels for containing the reaction mixture or cells and useful in thepresent invention will be apparent to the skilled artisan.

The addition of the components of the assay for detecting the activationstate or activity of an activatable element, or modulation of suchactivation state or activity, may be sequential or in a predeterminedorder or grouping under conditions appropriate for the activity that isassayed for. Such conditions are described here and known in the art.Moreover, further guidance is provided below (see, e.g., in theExamples).

As will be appreciated by one of skill in the art, the instant methodsand compositions find use in a variety of other assay formats inaddition to flow cytometry analysis. For example, a chip analogous to aDNA chip can be used in the methods of the present invention. Arrayersand methods for spotting nucleic acid to a chip in a prefigured arrayare known. In addition, protein chips and methods for synthesis areknown. These methods and materials may be adapted for the purpose ofaffixing activation state binding elements to a chip in a prefiguredarray. In some embodiments, such a chip comprises a multiplicity ofelement activation state binding elements, and is used to determine anelement activation state profile for elements present on the surface ofa cell.

In some embodiments, a chip comprises a multiplicity of the “second setbinding elements,” in this case generally unlabeled. Such a chip iscontacted with sample, preferably cell extract, and a secondmultiplicity of binding elements comprising element activation statespecific binding elements is used in the sandwich assay tosimultaneously determine the presence of a multiplicity of activatedelements in sample. Preferably, each of the multiplicity of activationstate-specific binding elements is uniquely labeled to facilitatedetection.

In some embodiments confocal microscopy can be used to detect activationprofiles for individual cells. Confocal microscopy relies on the serialcollection of light from spatially filtered individual specimen points,which is then electronically processed to render a magnified image ofthe specimen. The signal processing involved confocal microscopy has theadditional capability of detecting labeled binding elements withinsingle cells, accordingly in this embodiment the cells can be labeledwith one or more binding elements. In some embodiments the bindingelements used in connection with confocal microscopy are antibodiesconjugated to fluorescent labels, however other binding elements, suchas other proteins or nucleic acids are also possible.

In some embodiments, the methods and compositions of the instantinvention can be used in conjunction with an “In-Cell Western Assay.” Insuch an assay, cells are initially grown in standard tissue cultureflasks using standard tissue culture techniques. Once grown to optimumconfluency, the growth media is removed and cells are washed andtrypsinized. The cells can then be counted and volumes sufficient totransfer the appropriate number of cells are aliquoted into microwellplates (e.g., Nunc™ 96 Microwell™ plates). The individual wells are thengrown to optimum confluency in complete media whereupon the media isreplaced with serum-free media. At this point controls are untouched,but experimental wells are incubated with a modulator, e.g. EGF. Afterincubation with the modulator cells are fixed and stained with labeledantibodies to the activation elements being investigated. Once the cellsare labeled, the plates can be scanned using an imager such as theOdyssey Imager (LiCor, Lincoln Nebr.) using techniques described in theOdyssey Operator's Manual v1.2., which is hereby incorporated in itsentirety. Data obtained by scanning of the multiwell plate can beanalyzed and activation profiles determined as described below.

In some embodiments, the detecting is by high pressure liquidchromatography (HPLC), for example, reverse phase HPLC, and in a furtheraspect, the detecting is by mass spectrometry.

These instruments can fit in a sterile laminar flow or fume hood, or areenclosed, self-contained systems, for cell culture growth andtransformation in multi-well plates or tubes and for hazardousoperations. The living cells may be grown under controlled growthconditions, with controls for temperature, humidity, and gas for timeseries of the live cell assays. Automated transformation of cells andautomated colony pickers may facilitate rapid screening of desiredcells.

In some embodiments, the activation level of an activatable element ismeasured using Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Abinding element that has been labeled with a specific element binds tothe activatable element. When the cell is introduced into the ICP, it isatomized and ionized. The elemental composition of the cell, includingthe labeled binding element that is bound to the activatable element, ismeasured. The presence and intensity of the signals corresponding to thelabels on the binding element indicates the level of the activatableelement on that cell (Tanner et al. Spectrochimica Acta Part B: AtomicSpectroscopy, (2007), 62(3):188-195.).

Flow cytometry or capillary electrophoresis formats can be used forindividual capture of magnetic and other beads, particles, cells, andorganisms.

Flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.Customized tools, labware, and liquid, particle, cell and organismtransfer patterns allow different applications to be performed.Databases allow method and parameter storage. Robotic and computerinterfaces allow communication between instruments.

In some embodiment, the methods of the invention include the use ofliquid handling components. The liquid handling systems can includerobotic systems comprising any number of components. In addition, any orall of the steps outlined herein may be automated; thus, for example,the systems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which can be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates;automated lid or cap handlers to remove and replace lids for wells onnon-cross contamination plates; tip assemblies for sample distributionwith disposable tips; washable tip assemblies for sample distribution;96 well loading blocks; cooled reagent racks; microtiter plate pipettepositions (optionally cooled); stacking towers for plates and tips; andcomputer systems.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting to perform all steps of screening applications. This includesliquid, particle, cell, and organism manipulations such as aspiration,dispensing, mixing, diluting, washing, accurate volumetric transfers;retrieving, and discarding of pipet tips; and repetitive pipetting ofidentical volumes for multiple deliveries from a single sampleaspiration. These manipulations are cross-contamination-free liquid,particle, cell, and organism transfers. This instrument performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and high capacity operation.

In some embodiments, chemically derivatized particles, plates,cartridges, tubes, magnetic particles, or other solid phase matrix withspecificity to the assay components are used. The binding surfaces ofmicroplates, tubes or any solid phase matrices include non-polarsurfaces, highly polar surfaces, modified dextran coating to promotecovalent binding, antibody coating, affinity media to bind fusionproteins or peptides, surface-fixed proteins such as recombinant proteinA or G. nucleotide resins or coatings, and other affinity matrix areuseful in this invention.

In some embodiments, platforms for multi-well plates, multi-tubes,holders, cartridges, minitubes, deep-well plates, microfuge tubes,cryovials, square well plates, filters, chips, optic fibers, beads, andother solid-phase matrices or platform with various volumes areaccommodated on an upgradable modular platform for additional capacity.This modular platform includes a variable speed orbital shaker, andmulti-position work decks for source samples, sample and reagentdilution, assay plates, sample and reagent reservoirs, pipette tips, andan active wash station. In some embodiments, the methods of theinvention include the use of a plate reader.

In some embodiments, thermocycler and thermoregulating systems are usedfor stabilizing the temperature of heat exchangers such as controlledblocks or platforms to provide accurate temperature control ofincubating samples from 0° C. to 100° C.

In some embodiments, interchangeable pipet heads (single ormulti-channel) with single or multiple magnetic probes, affinity probes,or pipetters robotically manipulate the liquid, particles, cells, andorganisms. Multi-well or multi-tube magnetic separators or platformsmanipulate liquid, particles, cells, and organisms in single or multiplesample formats.

In some embodiments, the instrumentation will include a detector, whichcan be a wide variety of different detectors, depending on the labelsand assay. In some embodiments, useful detectors include a microscope(s)with multiple channels of fluorescence; plate readers to providefluorescent, ultraviolet and visible spectrophotometric detection withsingle and dual wavelength endpoint and kinetics capability,fluorescence resonance energy transfer (FRET), luminescence, quenching,two-photon excitation, and intensity redistribution; CCD cameras tocapture and transform data and images into quantifiable formats; and acomputer workstation.

In some embodiments, the robotic apparatus includes a central processingunit which communicates with a memory and a set of input/output devices(e.g., keyboard, mouse, monitor, printer, etc.) through a bus. Again, asoutlined below, this may be in addition to or in place of the CPU forthe multiplexing devices of the invention. The general interactionbetween a central processing unit, a memory, input/output devices, and abus is known in the art. Thus, a variety of different procedures,depending on the experiments to be run, are stored in the CPU memory.

These robotic fluid handling systems can utilize any number of differentreagents, including buffers, reagents, samples, washes, assay componentssuch as label probes, etc.

Gating

In another embodiment, a user may analyze the signaling insubpopulations based on surface markers. For example, the user couldlook at: “stem cell populations” by CD34+ CD38− or CD34+ CD33−expressing cells; drug transporter positive cells; e.g. P—P-glycoproteinpositive cells; or multiple leukemic subclones based on CD33, CD45,HLA-DR, CD11b and analyzing signaling in each subpopulation. In anotheralternative embodiment, a user may analyze the data based onintracellular markers, such as transcription factors or otherintracellular proteins; based on a functional assay (e.g., dye effluxassay to determine drug transporter+cells or fluorescent glucose uptake)or based on other fluorescent markers. In some embodiments, gates areused to identify the presence of specific subpopulations in existingindependent data. The existing independent data can be data stored in acomputer from a previous patient, or data from independent studies usingdifferent patients.

In some embodiments where flow cytometry is used, prior to analyzing ofdata the populations of interest and the method for characterizing thesepopulations are determined. For instance, there are at least two generalways of identifying populations for data analysis: (i) “Outside-in”comparison of Parameter sets for individual samples or subset (e.g.,patients in a trial). In this more common case, cell populations arehomogenous or lineage gated in such a way as to create distinct setsconsidered to be homogenous for targets of interest. An example ofsample-level comparison would be the identification of signalingprofiles in tumor cells of a patient and correlation of these profileswith non-random distribution of clinical responses. This is consideredan outside-in approach because the population of interest is pre-definedprior to the mapping and comparison of its profile to other populations.(ii) “Inside-out” comparison of Parameters at the level of individualcells in a heterogeneous population. An example of this would be thesignal transduction state mapping of mixed hematopoietic cells undercertain conditions and subsequent comparison of computationallyidentified cell clusters with lineage specific markers. This could beconsidered an inside-out approach to single cell studies as it does notpresume the existence of specific populations prior to classification. Amajor drawback of this approach is that it creates populations which, atleast initially, require multiple transient markers to enumerate and maynever be accessible with a single cell surface epitope. As a result, thebiological significance of such populations can be difficult todetermine. The main advantage of this unconventional approach is theunbiased tracking of cell populations without drawing potentiallyarbitrary distinctions between lineages or cell types.

Each of these techniques capitalizes on the ability of flow cytometry todeliver large amounts of multiparameter data at the single cell level.For cells associated with a condition (e.g. neoplastic or hematopoeticcondition), a third “meta-level” of data exists because cells associatedwith a condition (e.g. cancer cells) are generally treated as a singleentity and classified according to historical techniques. Thesetechniques have included organ or tissue of origin, degree ofdifferentiation, proliferation index, metastatic spread, and genetic ormetabolic data regarding the patient.

In some embodiments, the present invention uses variance mappingtechniques for mapping condition signaling space. These methodsrepresent a significant advance in the study of condition biologybecause it enables comparison of conditions independent of a putativenormal control. Traditional differential state analysis methods (e.g.,DNA microarrays, subtractive Northern blotting) generally rely on thecomparison of cells associated with a condition from each patient samplewith a normal control, generally adjacent and theoreticallyuntransformed tissue. Alternatively, they rely on multiple clusteringsand reclusterings to group and then further stratify patient samplesaccording to phenotype. In contrast, variance mapping of conditionstates compares condition samples first with themselves and then againstthe parent condition population. As a result, activation states with themost diversity among conditions provide the core parameters in thedifferential state analysis. Given a pool of diverse conditions, thistechnique allows a researcher to identify the molecular events thatunderlie differential condition pathology (e.g., cancer responses tochemotherapy), as opposed to differences between conditions and aproposed normal control.

In some embodiments, when variance mapping is used to profile thesignaling space of patient samples, conditions whose signaling responseto modulators is similar are grouped together, regardless of tissue orcell type of origin. Similarly, two conditions (e.g. two tumors) thatare thought to be relatively alike based on lineage markers or tissue oforigin could have vastly different abilities to interpret environmentalstimuli and would be profiled in two different groups.

When groups of signaling profiles have been identified it is frequentlyuseful to determine whether other factors, such as clinical responses,presence of gene mutations, and protein expression levels, arenon-randomly distributed within the groups. If experiments or literaturesuggest such a hypothesis in an arrayed flow cytometry experiment, itcan be judged with simple statistical tests, such as the Student'st-test and the X² test. Similarly, if two variable factors within theexperiment are thought to be related, the r² correlation coefficientfrom a linear regression is used to represent the degree of thisrelationship.

Classes of Cells

The activation state of an individual activatable element is either inthe on or off state. As an illustrative example, an individualphosphorylatable site on a protein will either be phosphorylated andthen be in the “on” state or it will not be phosphorylated and hence, itwill be in the “off” state. The terms “on” and “off,” when applied to anactivatable element that is a part of a cellular constituent, are usedhere to describe the state of the activatable element (e.g.,phosphorylated is “on” and non-phosphorylated is “off”), and not theoverall state of the cellular constituent of which it is a part.Typically, a cell possesses a plurality of a particular protein or otherconstituent with a particular activatable element and this plurality ofproteins or constituents usually has some proteins or constituents whoseindividual activatable element is in the on state and other proteins orconstituents whose individual activatable element is in the off state.Since the activation state of each activatable element is measuredthrough the use of a binding element that recognizes a specificactivation state, only those activatable elements in the specificactivation state recognized by the binding element, representing somefraction of the total number of activatable elements, will be bound bythe binding element to generate a measurable signal. The measurablesignal corresponding to the summation of individual activatable elementsof a particular type that are activated in a single cell is the“activation level” for that activatable element in that cell.

Activation levels for a particular activatable element may vary amongindividual cells so that when a plurality of cells is analyzed, theactivation levels follow a distribution. The distribution may be anormal distribution, also known as a Gaussian distribution, or it may beof another type. Different populations of cells may have differentdistributions of activation levels that can then serve to distinguishbetween the populations.

In some embodiments, the basis for classifying cells is that thedistribution of activation levels for one or more specific activatableelements will differ among different phenotypes. A certain activationlevel, or more typically a range of activation levels for one or moreactivatable elements seen in a cell or a population of cells, isindicative that that cell or population of cells belongs to adistinctive phenotype. Other measurements, such as cellular levels(e.g., expression levels) of biomolecules that may not containactivatable elements, may also be used to classify cells in addition toactivation levels of activatable elements; it will be appreciated thatthese levels also will follow a distribution, similar to activatableelements. Thus, the activation level or levels of one or moreactivatable elements, optionally in conjunction with levels of one ormore levels of biomolecules that may not contain activatable elements,of cell or a population of cells may be used to classify a cell or apopulation of cells into a class.

Once the activation level of intracellular activatable elements ofindividual single cells is known they can be placed into one or moreclasses. In some embodiments, cells are placed in predefined classes. Apredefined class encompasses a class of cells wherein every cell has thesame or substantially the same known activation level, or range ofactivation levels, of one or more intracellular activatable elements.For example, if the activation levels of five intracellular activatableelements are analyzed, predefined classes that encompass one or more ofthe intracellular activatable elements can be constructed based on theactivation level, or ranges of the activation levels, of each of thesefive elements. It is understood that activation levels can exist as adistribution and that an activation level of a particular element usedto classify a cell may be a particular point on the distribution butmore typically may be a portion of the distribution.

In addition to activation levels of intracellular activatable elements,expression levels of intracellular or extracellular biomolecules, e.g.,proteins, may be used alone or in combination with activation states ofactivatable elements to classify cells. Further, additional cellularelements, e.g., biomolecules or molecular complexes such as RNA, DNA,carbohydrates, metabolites, and the like, may be used in conjunctionwith activatable states or expression levels in the classification ofcells encompassed here.

In some embodiments, other characteristics that affect the status of acellular constituent may also be used to classify a cell. Examplesinclude the translocation of biomolecules or changes in their turnoverrates and the formation and disassociation of complexes of biomolecule.Such complexes can include multi-protein complexes, multi-lipidcomplexes, homo- or hetero-dimers or oligomers, and combinationsthereof. Other characteristics include proteolytic cleavage, e.g. fromexposure of a cell to an extracellular protease or from theintracellular proteolytic cleavage of a biomolecule.

A predefined class of cells, additionally, may be further divided intosubsets that are themselves predefined classes based on other factors,such as the expression level of extracellular or intracellular markers,nuclear antigens, enzymatic activity, protein expression andlocalization, cell cycle analysis, chromosomal analysis, cell volume,and morphological characteristics like granularity and size of nucleusor other distinguishing characteristics. For example, if B cellsrepresent a predefined class, they can be further subdivided based onthe expression of cell surface markers such as CD19, CD20, or CD22.

Alternatively, predefined classes of cells can be aggregated based uponshared characteristics that may include inclusion in one or moreadditional predefined class or the presence of extracellular orintracellular markers, similar gene expression profile, nuclearantigens, enzymatic activity, protein expression and localization, cellcycle analysis, chromosomal analysis, cell volume, and morphologicalcharacteristics like granularity and size of nucleus or otherdistinguishing characteristics.

The absence of a class is itself a predefined class; e.g., cells in asample may be classified as those belonging to a class and those notbelonging to that class, where the latter is itself considered a class.This is useful when it is desired to determine what the percentage ofthe total number of cells belong to one particular class.

The predefined classes may be determined empirically based on data fromindividuals that indicates status, e.g., health status. E.g., bloodsamples from the clinic and/or from clinical trials may be analyzedretrospectively to determine classes of cells; certain classes orquantitative features of the classes may be associated with certainknown outcomes for the patients. For example, blood samples may beobtained from cancer patients over the course of treatment. Variousoutcomes, from complete remission for a number of years, to death fromcancer or cancer recurrence after treatment, may be recorded. Profilesof the states of activatable elements in single cells, with or withoutmodulator, may be obtained from retrospective samples to determineclasses of cells present in the samples, numbers of cells in each class,relative numbers of class vs. class, and the like. These classes are“predefined” classes as that term is used herein, and the classes,together with their predictive value for various health statuses, may beplaced in a database that is then used for analysis of further samples.As more samples are obtained and correlated health status determined,the database may be modified.

Thus, in some embodiments, the invention encompasses a database ofclasses of cells, where the cells are classified at least in partaccording to the activation level of one or more activatable elements,and clinical outcomes for patients from whom the cells are derived. Sucha database may be on a computer-readable medium.

a. Rare Cells

In some embodiments, the cells are classified into a class that isconsidered a class of rare cells. In some embodiments, the presence ofrare cell populations is used to make a diagnosis, prognosis or topredict response to a treatment. The term “rare” as used herein is usedto denote a low numbers of abundance, uncommon, or scarce cells. It iscontemplated that the detection of rare cell populations can be used topredict changes in health status.

In some embodiments, the cells are classified as rare cells at least inpart according to the activation level of one or more activatableelements. The term “rare” as used herein designates cells of interestthat are to be detected. This term is not intended to limit the relativeabundances of the designated cell types, although it is preferable forthe rare cells to have a relative abundance of less the 25%, 10%, 5%,1%, 0.5%, and less.

Whether a particular cell is a rare cell can be viewed different ways.In a first manner of characterizing a cell as rare, the rare cell can besaid to be any cell that does not naturally occur as a significantfraction of a given sample. For example, for human or mammalian blood, arare cell may be any cell other than a subject's blood cell (such as anormal red blood cell and a normal white blood cell). In this view,cancer or other cells present in the blood would be considered rarecells. In addition, infiltrating cancer cells in a tissue should beconsidered rare cells. A second manner of characterizing a cell as raremight take into account the frequency with which that cell appears in asample or the frequency with respect to other cells. A cell can beconsidered rare when the frequency of the cell is compared to more thanone class of cells. When the rare cells are associated with apathological state such as cancer, the frequency of the rare cellpopulation can be compared to normal cells or to other cells associatedwith the pathological state. For example, a rare cell may be a cell thatappears at a frequency of approximately 1 to 50 cells per ml of blood. Arare cell may be present in a sample, blood or tissue in a concentrationof less than 1 in 10,000 cells, 1 in 100,000 cells, 1 in 1,000,000cells, 1 in 10,000,000 cells, 1 in 100,000,000 cells, or 1 in1,000,000,000 cells. Alternatively, rare cell frequency within a givenpopulation containing non-rare cells or other rare cells can include,but is not limited to, frequencies of less than about 1 cell in 100cells; 1 cell in 1,000 cells; 1 cell in 10,000 cells; 1 cell in 100,000cells; 1 cell in 1,000,000 cells; 1 cell in 10,000,000 cells; 1 cell in100,000,000 cells; or 1 cell in 1,000,000,000 cells.

In a third manner of characterizing a cell as rare, the rare cell can besaid to be a cell located at a different position when compared tonormal cells in a contour or density plot. The contour or density plotrepresents the number of cells that share a characteristic such as theactivation level of activatable proteins in response to a modulator. Forexample, when referring to activation levels of activatable elements inresponse to one or more modulator, normal individuals and patients witha pathological state might show populations with increased activationlevels in response to the one or more modulators. However, the number ofcells that have a specific activation level (e.g. specific amount of anactivatable element) might be different between normal individuals andpatients with a pathological state. Thus, a rare cell is a cell that iswithin a given region in the contour or density plot that is differentfrom the regions of normal cells. Rare cell frequency when compared todifferent regions containing non-rare cells or other rare cells caninclude, but is not limited to, a frequency of less than about 1 cell in10 cells, 1 cell in 20 cells, 1 cell in 50 cells, 1 cell in 100 cells, 1cell in 1,000 cells, 1 cell in 100,000 cells; or 1 cell in 1,000,000cells. The frequency of rare cells within a region can be determined byusing mathematical estimates of the centers of the contour or densityplot, densities within the blobs in a plots, or the relative position ofeach blob in the plot to each other in N-space define the placements.For example, the frequency of the rare cell population within a regioncan be determined by using an eigenvector approach. Another way tocalculate the frequency of the rare cell population within a region isto describe the surface of the density and calculate the differences inthe volumes (e.g. how much does one shape protrude from the other). Insome embodiments, the individual status of an individual (e.g. clinicaloutcome) is determined when the number of rare cells within a region ishigher that a threshold number. In some instances, the threshold numberis 0 and the finding of 1 rare cell within a region would indicate of astatus of the individual (e.g. a cancer cell is present and treatmentmust begin). In other instances, the threshold number is 1. In stillother instances, the threshold number is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700,800, 900, or 1000 cells.

The methods of the present invention allows for the determination of thestatus of an individual (e.g. a clinical outcome) by detecting rarecells at lower relative abundances. For example, a diagnosis can be madein a patient by detecting a rare population of cells associated with apathological state such as cancer. In some embodiments, the status of anindividual (e.g. a clinical outcome) can be determined when the numberof rare cells is fewer than 10⁻² to 10⁻⁴ cells (one rare cell in 100 to10,000 total cells). For example, the presence of 1×10⁻², 1×10⁻³,2×10⁻³, 3×10⁻³, 4×10⁻³, 5×10⁻³, 6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³, 10×10⁻⁴,2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁻⁴, 6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or 9×10⁻⁴ rarecells is used to determine the status of an individual (e.g. a clinicaloutcome such as probability of relapse). In some embodiments, the numberof rare cells used to determine the status of an individual is fewerthan 1×10⁻². In some embodiments, the number of rare cells used todetermine the status of an individual is fewer than 5×10⁻⁴. In someembodiments, the number of rare cells used to determine the status of anindividual is fewer than 4.5×10⁻⁴. In some embodiments, the number ofrare cells used to determine the status of an individual is fewer than4×10⁻⁴. In some embodiments, the number of rare cells used to determinethe status of an individual is fewer than 3.5×10⁻⁴. In some embodiments,the number of rare cells used to determine the status of an individualis fewer than 3.5×10⁻⁴. In some embodiments, the number of rare cellsused to determine the status of an individual is fewer than 2×10⁻⁴.

In some embodiments, the methods describe herein provide for trackingthe emergence and/or disappearance of rare cell populations. In someembodiments, the methods described herein provides for the determinationof the presence or absence of pre-existing populations of rare cells asis the case when a patient is originally diagnosed with a condition suchas cancer. These pre-existing cells can be from a single clone of cellsor multiple clones. In some embodiments, the methods described hereinprovides for the determination for the presence or absence of rare cellspopulation that develops over time such as a rare cell population thatdevelops over the course of a treatment. These later developed cells canbe from a single clone of cells or multiple clones. Thus, in someembodiments, the methods described herein provide for the determinationof one or more rare cell population at diagnosis, during treatment andafter treatment. The methods described herein provide for the monitoringof a patient at several stages, thus, allowing for example theidentification of rare cells populations that have responded totreatment, rare cell population that did not respond and/or rare cellspopulations that emerge during the course of treatment or duringremission stages. The determination of rare cells populations allows forvery sensitive detection of changes in the health status of anindividual, which allows for early diagnosis and/or treatment.

The methods of the present invention allows for the determination of thestatus of an individual (e.g. a clinical outcome) by detecting rarecells that are strongly associated with said status. In someembodiments, the p value in the analysis is below 0.05, 04, 0.03, 0.02,0.01, 0.009, 0.005, or 0.001. In some embodiments, the p value is below0.001. Thus in some embodiments, the status of an individual can bedetermined by detecting rare cells wherein the p value is below 0.05,04, 0.03, 0.02, 0.01, 0.009, 0.005, or 0.001. In some embodiments, the pvalue is below 0.001. In some embodiments, the status of an individualcan be determined by detecting rare cells wherein the AUC value ishigher than 0.5, 0.6, 07, 0.8 or 0.9. In some embodiments, the status ofan individual can be determined by detecting rare cells wherein the AUCvalue is higher than 0.7. In some embodiments, the status of anindividual can be determined by detecting rare cells wherein the AUCvalue is higher than 0.8. In some embodiments, the status of anindividual can be determined by detecting rare cells wherein the AUCvalue is higher than 0.9.

Quantitative Analysis of Predefined Classes

Once a sufficient number of single cells have been placed into classesof cells (e.g. predefined classes of cells), the status of an individual(e.g. health status) can be determined by performing a quantitativeanalysis on one or more the predefined classes of cells. In someembodiments, the minimum number of single cells in a plurality of cellsthat is examined in order to determine an individual's health status isabout 10, 100, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000,1,000,000, 2,500,000, 5,000,000, or 10,000,000 cells. In some examples,the method of the present invention can be used to detect less than 200cells in a sample for determining a health status of an individual.

In some embodiments, the maximum number of single cells in a pluralityof cells that is examined in order to determine an individual's healthstatus is about 10, 100, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000,500,000, 1,000,000, 2,500,000, 5,000,000, or 10,000,000 cells.

Any suitable method of quantitative analysis can be used including, butnot limited to quantifying the number of cells in a particular class,determining if the number of cells in a particular predefined class isgreater than, equal to, or less than a threshold number, determining theratio of number of cells in one or more predefined classes to number ofcells in one or more other predefined classes, determining the if theratio of one or more predefined classes of compared to one or more otherpredefined classes of cells is greater than, equal to or less than athreshold number. If sequential samples are obtained, thendeterminations of the rate of change in the number of cells inpredefined classes or ratios of numbers of cells can be calculated.

In the simplest quantitative analysis, the number of cells in one ormore classes is compared to a threshold number, where if the number ofcells in the predefined class is greater than, equal to, or less thanthe threshold number, the status of the individual may be determined.

In certain instances, the finding of 0 cells in a predefined class maybe determinative as to an individual's status. In this case, thethreshold number is 1, and finding fewer than one cell is indicative ofthe status of the individual. For example, if a predefined class ofcells is associated with the presence or recurrence of a disease, forexample, cancer, then the finding of 0 cells in the predefined class ofcells provides evidence that the individual does not have the disease orhas not experienced a recurrence.

In some embodiments, the presence of 1 cell in a predefined class may bedeterminative of an individual's status. In this case, the thresholdnumber is 0, and finding even a single cell (more than zero) isindicative of the status of the individual. In an individual with highrisk of developing a disease, where pre-pathologic and/or pathologiccells belong to a signature predefined class of cells, the finding of 1cell in this predefined class indicates that the in the case of apre-pathological condition, the disease process has begun, or, in thecase of a pathological condition, the individual is already afflicted,but may be yet to manifest disease symptoms. Even in otherwise healthyindividuals, the appearance of a single cell of a particular stateindicates that pathology or disease is present. For example, theappearance in a blood sample of a single cell in a predefined classknown to be that of a certain category of cancer indicates the presenceof such a cancer, whether or not other findings indicate any diseasepresence. Such a finding would allow early treatment, that may be lesstoxic and/or be associated with a greater degree of disease control orcure. In some embodiments, the appearance of one cell in two or moredifferent predefined classes indicates a particular disease status. Insome embodiments, the minimal status of a pathological state isdetermined by a finding of even a single cell.

In some cases, the number of cells in a predefined class may bedeterminative of an individual's status only if the number exceeds or isless than a certain threshold number of cells. For example, a thresholdnumber may represent a clinically observed dividing line, associatedwith patient outcome. Individuals above the threshold may have a worseprognosis than those below the threshold number and may require moreimmediate and/or more aggressive treatment than individuals below thethreshold number. The threshold number can be theoretically, or, moretypically, empirically derived, e.g., from retrospective analysis ofclinical samples as described herein. In some instances, the thresholdnumber is 0 and the finding of cells in the predefined class wouldindicate that the status of the individual has changed and treatmentmust begin. In other instances, the threshold number is 1. In stillother instances, the threshold number is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700,800, 900, 1000, 5,000, 10,000, 100,000, or 1,000,000 cells.

In some embodiments, the number of cells that will be determinative ofthe individual status will depend on the phenotype of the cells in thepredefined class. For example, in determining the probability of relapsein cancer patients, patients that have cells associated with a malignantphenotype would have relapses if they have number of cells in thepredefined class higher than for example 10⁻⁵, whereas patients withcells associated with a less malignant phenotype would have relapses ifthey have number of cells in a predefined class higher than for example10⁻².

In other embodiments, rather than a threshold number, the finding of acertain number of cells in a particular class in a sample from anindividual may be correlated with a certain probability of a particularstatus for the individual. For example the presence of about 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300,400, 500, 600, 700, 800, 900, 1000, 5,000, 10,000, 100,000, or 1,000,000cells in a predefined class may be indicative of an individual's status.Ranges of cell numbers for a given condition of sampling (e.g., numberof cells per 5 or 10 ml blood draw) are useful. Ranges may be any usefulrange that has been correlated to a particular outcome or status, andmay be a minimum of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900,1000, 5,000, 10,000, 100,000, or 1,000,000 cells and a maximum of about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5,000, 10,000,100,000, 1,000,000 or 10,000,000 cells. For example, for a blood drawunder certain defined conditions (e.g., a blood draw of a particularvolume, or normalized to a particular volume) which contains a certainnumber of cells in a predefined class, may indicate that an individualis at a certain percentage of risk for developing a certain conditionwithin a given time. As an example only, the presence of 10-100 cells ofa certain predefined class in a blood draw of 10 ml may be associatedwith a 50% probability of pathology occurring within 5 years. It will beappreciated that ranges and probabilities may be adjusted as databasesbecome more extensive.

In some embodiments, the number of cells in a predefined class may bedeterminative when the number of cells is fewer than 10⁻³ to 10⁻⁴ cells(one cell in the predefined class in 1,000 to 10,000 total cells). Forexample, the presence of 1×10⁻³, 2×10⁻³, 3×10⁻³, 4×10⁻³, 5×10⁻³, 6×10⁻³,7×10⁻³, 8×10⁻³, 9×10⁻³, 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁻⁴, 6×10⁻⁴,7×10⁴, 8×10⁻⁴, or 9×10⁻⁴ cells in a predefined class may be indicativeof an individual's status. In some embodiments, the number of cells in apredefined class maybe determinative when the number of cells is fewerthan 5×10⁻⁴ In some embodiments, the number of cells in a predefinedclass maybe determinative when the number of cells is fewer than4.5×10⁻⁴. In some embodiments, the number of cells in a predefined classmaybe determinative when the number of cells is fewer than 4×10⁻⁴. Insome embodiments, the number of cells in a predefined class maybedeterminative when the number of cells is fewer than 3.5×10⁻⁴. In someembodiments, the number of cells in a predefined class maybedeterminative when the number of cells is fewer than 3.5×10⁻⁴. In someembodiments, the number of cells in a predefined class maybedeterminative when the number of cells is fewer than 2×10⁻⁴.

In some embodiments, the number of cells in a predefined class may bedeterminative when the number of cells is higher than 10⁻² to 10⁻⁴ cells(one cell in the predefined class in 100 to 10,000 total cells). Forexample, the presence of 1×10⁻², 2×10⁻², 3×10⁻², 4×10⁻², 5×10⁻², 6×10⁻²,7×10⁻², 8×10⁻², 9×10⁻², 1×10⁻³, 2×10⁻³, 3×10⁻³, 4×10⁻³, 5×10⁻³, 6×10⁻³,7×10⁻³, 8×10⁻³, 9×10⁻³ or 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁻⁴,6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or 9×10⁻⁴ cells in a predefined class may beindicative of an individual's status. In some embodiments, the number ofcells in a predefined class maybe determinative when the number of cellsis higher than 5×10⁻⁴. In some embodiments, the number of cells in aredefined class maybe determinative when the number of cells is higherthan 4.5×10⁻⁴. In some embodiments, the number of cells in a predefinedclass maybe determinative when the number of cells is higher than4×10⁻⁴. In some embodiments, the number of cells in a predefined classmaybe determinative when the number of cells is higher than 3.5×10⁻⁴. Insome embodiments, the number of cells in a predefined class maybedeterminative when the number of cells is higher than 3.5×10⁻⁴. In someembodiments, the number of cells in a predefined class maybedeterminative when the number of cells is higher than 2×10⁻⁴.

In some embodiments, the number of cells in a predefined class may bedeterminative when it is correlated with a predetermined clinicalparameter. For example in determining the probability of relapse in AMLpatients, patients that have a favorable cytogenetic subtype would haverelapses if they have number of cells in a predefined class higher thanfor example 10⁻², whereas patients with adverse cytogenetic subtypeswould have relapses if they have number of cells in a predefined classhigher than for example 10⁻⁴. In other diseases, the presence of evenone cell in a predefined class may indicate a relapse.

When a series of samples is taken over time, a predefined class of cellscan be analyzed to see if it is increasing or decreasing in number at arate that will cause the predefined class of cells to cross a thresholdnumber in the future. FIG. 1 illustrates this situation; in this case, aseries of samples is analyzed and at a certain point the number of cellsin a particular class crosses a threshold indicating a change in status.By predicting when an individual may cross a threshold number, earlieraction may be taken to either prevent the crossing of the thresholdnumber in cases where crossing is associated with a detrimental outcome,or accelerate the crossing of the threshold number where crossing isassociated with a better outcome or prognosis. For example, if the trendshows that a particular predefined class of cells in patient associatedwith the relapse of disease will cross the threshold number in a month,prophylactic treatment can be initiated to prevent the occurrence.

In some cases, the rate of change of the number of cells in a predefinedclass may be an indicator of present or future health status. This maybe combined with absolute numbers, or used as a further indicator withan absolute number. This is similar to the situation with PSA, where alow absolute number is generally taken as a sign of healthy prostate,but if an increase is seen over a series of samples further testing isindicated, even if each individual number is in itself not indicative ofpathology. As with threshold values or ranges, certain rates of change,or ranges of rates of change, may be associated with certainprobabilities of outcome; such probabilities may be modified based onthe absolute number of cells in the predefined class; e.g., a low rateof change couple with high absolute numbers may indicate the sameprobability of a given outcome or health status as a high rate of changecoupled with low absolute numbers.

In some embodiments of the invention, a series of samples is taken froman individual undergoing treatment for a condition, e.g., treatment fora cancer. The samples may be evaluated for the number of cells thatcorrelate with the cancer, and the rate of change in numbers of thesecells during treatment may be correlated with a particular outcome;e.g., a rapid decrease in cancer cell number may indicate a morepositive prognosis than a less rapid decrease; in addition, changes inthe rate of change (e.g., rapid decrease followed by little or nodecrease) also may have prognostic value. Such evaluations of the rateof change during treatment may be combined with numbers of cells in oneor more predefined classes at the conclusion of treatment, and/or aftertreatment, to further refine the prognostic and diagnostic accuracy.

The threshold number for a particular predefined class may differ basedon sample location. For example, cells isolated from peripheral bloodand those from bone marrow or lymph nodes may have their own clinicallysignificant threshold numbers for specific predefined classes of cells.Ratios and other mathematical methods of comparison may be developed toallow the comparison of cells isolated from different bodily locations,thereby providing greater flexibility to the clinician in procuring asample of a plurality of cells.

When more than one predefined class of cells are present, comparativequantitative analyses can be performed to determine an individual'sstatus (e.g., health status). Numerous comparative and statisticaltechniques are known in the arts for the analysis of different groups.Examples of such statistical methods include but are not limited toX²-test, Student T test, Mann-Whitney U test, log-rank, Breslow test,Kaplan, Meier, Spearman's rank correlation, logistic regression model,Cox models, or AUC plots. In some embodiments, the p value in theanalysis is below 0.05, 04, 0.03, 0.02, 0.01, 0.009, 0.005, or 0.001. Insome embodiments, the p value is below 0.001. Thus in some embodiments,the status of an individual can be determined by performing aquantitative analysis on one or more predefined classes of cells whereinthe p value is below 0.05, 04, 0.03, 0.02, 0.01, 0.009, 0.005, or 0.001.In some embodiments, the p value is below 0.001. In some embodiments,the status of an individual can be determined by performing aquantitative analysis on one or more predefined classes of cells whereinthe AUC value is higher than 0.5, 0.6, 07, 0.8 or 0.9. In someembodiments, the status of an individual can be determined by performinga quantitative analysis on one or more predefined classes of cellswherein the AUC value is higher than 0.7. In some embodiments, thestatus of an individual can be determined by performing a quantitativeanalysis on one or more predefined classes of cells wherein the AUCvalue is higher than 0.8. In some embodiments, the status of anindividual can be determined by performing a quantitative analysis onone or more predefined classes of cells wherein the AUC value is higherthan 0.9.

In some embodiments, the number of cells in one predefined class can becompared to the number of cells in another predefined class by takingthe ratio of the two. FIG. 2 illustrates a situation in which cells arequantitated in a number of different predefined classes; FIG. 2B showsvarious exemplary ratios that could be obtained. Alternately, the numberof cells in one predefined class can be compared by taking the ratio ofthis class and the cell number from a combination of predefined classes.As a simple example, if predefined class 1 has 200 cells and predefinedclass 2 has 1000 cells, the ratio of cells in A to cells in B would be0.2, or 20%.

The simplest ratio is the ratio of one predefined class of cells tototal cells. In this case, the term “total cells” includes all cells inthe sample, total cells collected for analysis or total live cellsanalyzed, whether or not their status, e.g., the activation level oftheir intracellular activatable elements, has been determined. Thus,“total cells” includes a predefined class that encompasses the total ofany cell in the sample. In some embodiments, the ratio is that of onepredefined class to total cells of a certain type, e.g., totalleukocytes, or total cells with a particular set of cell surfacemarkers.

FIG. 15 is a diagram showing the method of determining a status of anindividual (e.g. health status) at a certain stage, In some embodiments,the method of the present invention can be applied to an individualbefore a diagnosis, an individual undergoing a treatment, or anindividual in remission or having a relapse as depicted in step 1500 ofFIG. 15. In step 1501, cells from the individual are analyzed accordingto the method described herein. In some embodiments, one or more samplesare taken from the individual, and subjected to a modulator, asdescribed herein. In some embodiments, the sample is divided intosubsamples that are each subjected to a different modulator. Aftertreatment with the modulator, single cells in the sample or subsampleare analyzed to determine the activation level of one or moreactivatable elements. Any suitable form of analysis that allows adetermination of cell activation level(s) may be used. In someembodiments, the analysis includes the determination of the activationlevel of an intracellular element, e.g., a protein. In some embodiments,the analysis includes the determination of the activation level of anactivatable element, e.g., an intracellular activatable element such asa protein, e.g., a phosphoprotein. The analysis of activation level ofan intracellular element, e.g., a protein, may be achieved by the use ofactivation state-specific binding elements, such as antibodies, asdescribed herein. A plurality of activatable elements may be examined.

In step 1501 of FIG. 15, cells are analyzed by determining the number ofcells, cell ratio or rate of change. The analysis can be performed byany method described herein such as the method described in FIGS. 1 to4. In some embodiments, the p value is below 0.05, 04, 0.03, 0.02, 0.01,0.009, 0.005, or 0.001. In some embodiments, the p value is below 0.001.In some embodiments, the AUC value is higher than 0.5, 0.6, 07, 0.8 or0.9. In some embodiments, the AUC value is higher than 0.8.

In step 1502 of FIG. 15, a diagnosis, prognosis, method of treatment orresponse to treatment is determined after the analysis in step 1501.Thus the analysis of step 1501 allows for the diagnosis, prognosis,choice or modification of treatment, and/or monitoring of a disease,disorder, or condition. In some embodiments, the determination of thestatus of an individual can be determining whether the individual is inthe normal range for a particular condition or whether the individualhas a pre-pathological or pathological condition warranting monitoringand/or treatment. In some embodiments, the determination of the statusof an individual can be determining the minimal status of a pathologicalstate. The determination of the status may also indicate response of anindividual to treatment for a condition. In some embodiments, thedetermination of the status of an individual may be used to ascertainwhether a previous condition or treatment has induced a newpre-pathological or pathological condition that requires monitoringand/or treatment. In another embodiment, the status of an individual canindicate an individual's predicted or actual response to treatment for apre-pathological or pathological condition. In some embodiments, theanalysis obtained in step 1501 may be used to determine the best therapyfor an individual, which may include the determination that the besttherapy for a patient is supportive care. In a further embodiment, thestatus of an individual may indicate an individual's immunologic statusand may reflect a general immunologic status, an organ or tissuespecific status, or a disease related status.

It will be appreciated that further ratios are possible. Thecombinations are limited only by the number of classes present in thesample. It will also be appreciated that databases may be constructedfor all such ratios, and that any such ratio that has a correlation withthe status of an individual may be used in the methods of the invention.

Ratios may be used alone, or in combination with numbers of cells insingle classes, to provide an indication of the status of theindividual. Thus, all analyses described herein for threshold analysis,rate of change analysis, absolute number analysis, or combinationsthereof, also apply to ratios of cells.

In some embodiments, a ratio of about 0, 0.0000001%, 0.000001%,0.00001%, 0.0001%, 001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, 5.0%,10%, 20%, 40%, 60%, 80%, 90%, 95%, or 100% can be determinative of anindividual's status. In other embodiments, whether the calculated ratiolies above or below a threshold ratio is also determinative. Thethreshold ratio may be about 0, 0.0000001%, 0.000001%, 0.00001%,0.0001%, 001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, 5.0%, 10%, 20%,40%, 60%, 80%, 90%, 95%, or 100%. For example, in some embodiments, theexistence of minimal residual disease after treatment may be when theratio of the number of cells exhibiting a cancerous state to total cellsin a sample, e.g., a blood sample, exceeds a certain percentage, such as0.0001%, 0.001%, 0.01%, or 0.1%.

As with absolute numbers, it will often be useful to correlate a ratioof predefined classes with a probability of an outcome; in someembodiments, a range of ratios may be correlated with a probability.Such a range may be from a minimum of 0, 0.0000001%, 0.000001%,0.00001%, 0.0001%, 001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, 5.0%,10%, 20%, 40%, 60%, 80%, 90%, 95% to a maximum of 0.0000001%, 0.000001%,0.00001%, 0.0001%, 001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, 5.0%,10%, 20%, 40%, 60%, 80%, 90%, 95%, or 100%.

In some embodiments, where multiple samples are available sequentiallyover time from the same location, the ratio between particularpredefined classes of cells can be analyzed to see if the ratio istrending in a particular direction, just as for absolute numbers. FIG. 3illustrates such an analysis. If the ratio appears that it may cross athreshold ratio in the future, prophylactic treatment or other desirablecourse of action can be taken to prevent or accelerate the ratio fromcrossing the threshold ratio.

When sequential samples are available, a predefined class of cells canalso be analyzed by measuring the rate of change in the cell numberwithin the class (see FIG. 4). One common measurement of the rate ofchange is the doubling time of the number of cells in a predefinedclass. When data from multiple predefined classes is available overtime, the rate of change in the ratio between the classes can also bemeasured.

In some embodiments, the rate of activation or deactivation of aparticular intracellular activatable element with a specific modulatoror class of modulators may define a predefined class. The activationrate/deactivation rate can be determined through sequential measurementson cells obtained at different time points from the same source orlocation. Alternatively, the activation/deactivation rate can bedetermined from a plurality of cells that are obtained at the same time,but are assayed over time.

While some embodiments are associated with placing single cells inpredefined classes, in other embodiments, the appearance of one or morecells outside the predefined classes may be indicative of significantchanges in the status of an individual. Of particular interest indetermining the status of an individual is the detection and analysis ofclasses of cells that have one or more different activation levelscompared to normal control values, or to previous determinations madefrom a sample from the individual. The different activation levels canbe the result of the disappearance of one or more previous identifiedactivation levels from one or more predefined classes of cells.Alternatively, the different activation levels may be the result of theappearance of a new, activation level. The analysis of cells with one ormore different activation levels is the same as for other classes ofcells, but cells that have deletions of or additions to previouslyidentified activation levels are often of greater clinical significance.For example, a hallmark of cancer is genomic instability. The appearanceof a class of cells with one or more different activation levels duringthe course of cancer treatment may signify that a mutation has occurredand a new clonal population has arisen. Mutations in such instances arefrequently associated with increased resistance to the employedtreatment agents and such cells often comprise a major portion of thecancerous cells when a patient experiences a recurrence.

In the determination of the status of an individual along a healthcontinuum, other factors can be considered. Any factor that givesadditional predictive or diagnostic power to the single cell analysesdescribed herein may be used. Such factors are well-known in the art.These include an individual's gender; race; current age; age at the timeof disease presentation; age at the time of treatment; clinical stage ofdisease; genetic results, number of previous therapies; type of previoustherapies; response to previous therapy or therapies; time from lasttreatment; blood cell count; bone marrow reserves; and performancestatus, patient's past medical history, family history of any medicalproblems, patient's social history, as well as any current medicalhistory termed “review of systems”, and physical exam findings. Otherfactors are more specific to the specific condition being evaluated,e.g., percentage of blasts in bone marrow as an indicator of certainleukemias. Such factors are well-known in the art for particulardiseases and conditions. Examples of tests that can be performedtogether with the methods described herein include, but are not limitedto, immunophenotyping, morphology, conventional cytogenetics, molecularcytogenetics, molecular genetics and HLA typing.

Status of the Individual

The techniques and methods of this invention allow for the determinationof the status of an individual for any condition for which a correlationbetween the condition, its prognosis, course of treatment, or otherrelevant characteristic, and the state of single cells, e.g., activationlevel of one or more activatable elements, in samples from individualsmay be ascertained. In some embodiments, samples are blood samples andconditions that may be examined using the techniques of the inventionare those that cause alterations in single cells found in blood samples.However, the invention is not limited to the use of blood samples, andany condition which leads to a change in single cells in an area of theindividual amenable to sampling may be examined by the techniques of theinvention.

In some embodiments, the invention provides a method of predicting achange in a health status in an individual from a first state to asecond state comprising: determining the presence of a first and secondclass of cells in a sample from the individual, the presence beingdetermined by a method comprising determining an activation level of anintracellular activatable element in single cells from said sample,classifying single cells into the first and second class, wherein atleast one class is classified based on the activation level; calculatinga ratio of the first and second class of cells and using the ratio topredict said change in health status; and predicting a change in ahealth status in the individual from a first state to a second statewhen said ratio exceeds a threshold number. In some embodiments, thethreshold number expressed as a percentage is 30%. In some embodiments,the threshold number expressed as a percentage is 5%. In someembodiments threshold number expressed as a percentage is 1%. In someembodiments, the threshold number expressed as cell frequency is 10⁻².In some embodiments, the threshold number expressed as cell frequency is10⁻³. In some embodiments, the threshold number expressed as cellfrequency is 10⁻⁴.

In some embodiments, the health status or the predicted health status ofan individual places the individual along a health continuum thattypically runs from a normal or healthy state to one or morepre-pathologic states, and finally to a pathologic state. In someinstances, the health continuum may run from a normal state to apathological state without an intervening pre-pathologic state. Thehealth continuum may also comprise a partial continuum of theaforementioned states or a portion of one state. The health continuummay be related to the general health status of an individual, an organor organ system or the individual component tissues of an organ.Additionally, the health continuum may be specific for a family ofrelated diseases or disorders, a particular disease or disorder orsubtypes of a disease or disorder.

In some embodiments, an individual to be evaluated has not beendiagnosed with a pre-pathologic or pathologic condition but isundergoing a screening. In some embodiments, the minimal status of apathological state is determined. In certain instances, the finding of 0cells associated with a pathological state may be determinative as tominimal status of a pathological state. For example, the finding of 0cells associated with a pathological state provides evidence that theindividual does not have the pathological state or has not experienced arecurrence. In some embodiments, the presence of 1 cell associated witha pathological state may be determinative of an individual's status. Inthis case, the threshold number is 0, and finding even a single cell(more than zero) is indicative of the minimal status of the pathologicalstate. For example, the finding of 1 cell that is associated with ahighly malignant cancer phenotype indicates that the in the case ofcancer, the disease process has begun, but may be yet to manifestdisease symptoms. In an individual who has been treated for thepathological condition, the detection of cells associated with thepathological state indicates that treatment is incomplete. In otherinstances, a finding of a number higher than a threshold of cellsassociated with a pathological state may be determinative of anindividual's status. For example, a finding of equal or higher that 10⁻⁴cells associated with a cancer phenotype may indicate that theindividual is at risk of having a relapse, whereas a finding of lessthan 10⁻⁴ cells may indicate that the individual is at very low risk ofrelapse. The minimal status of a pathological state can thus distinguishwho needs intensive and potentially more toxic therapy from those who donot. In some cases the minimal status may also inform on the timing of aclinical intervention.

In these embodiments, one or more samples may be taken from theindividual, and subjected to a modulator, as described herein. In someembodiments, the sample is divided into subsamples that are eachsubjected to a different modulator. After treatment with the modulator,single cells in the sample or subsample are analyzed to determine theiractivation level(s). Any suitable form of analysis that allows adetermination of cell activation level(s) may be used. In someembodiments, the analysis includes the determination of the activationlevel of an intracellular element, e.g., a protein. In some embodiments,the analysis includes the determination of the activation level of anactivatable element, e.g., an intracellular activatable element such asa protein, e.g., a phosphoprotein. Determination of the status may beachieved by the use of activation state-specific binding elements, suchas antibodies, as described herein. A plurality of activatable elementsmay be examined. Single cells may be placed into predefined classes, andthe status of the individual determined based on the classes into whichcells are categorized. In some embodiments, a quantitative analysis ofthe number of cells in one or more classes is performed to determine thestatus of the individual. In some embodiments, the status to bedetermined includes the emergence of a new pre-pathologic or pathologiccondition, including a malignancy. Diagnosis, prognosis, and/or a courseof treatment may also be determined based on the analysis of the classesof cells. In some embodiments, the p value in the analysis is below0.05, 04, 0.03, 0.02, 0.01, 0.009, 0.005, or 0.001. In some embodiments,the p value is below 0.001. In some embodiments, the AUC value is higherthan 0.5, 0.6, 07, 0.8 or 0.9. In some embodiments, the AUC value ishigher than 0.8.

In some embodiments, an individual to be evaluated has already beensubjected to at least one form of treatment for a condition, e.g., amalignancy. In some embodiments, the invention provides methods of thedetermination of the minimal residual status of disease in an individualwho has received treatment. In these embodiments, one or more samplesmay be taken from the individual, and subjected to one or moremodulators, as described herein. In some embodiments, the sample isdivided into subsamples that are each subjected to one or more differentmodulators. After treatment with one or more modulators, single cells inthe sample or subsample are analyzed to determine their activationlevel(s). Any suitable form of analysis that allows a determination ofcell activation level(s) may be used. In some embodiments, the analysisincludes the determination of the activation level of an intracellularelement, e.g., a protein. In some embodiments, the analysis includes thedetermination of the activation level of an activatable element, e.g.,an intracellular activatable element such as a protein, e.g., aphosphoprotein. Determination of the status may be achieved by the useof activation state-specific binding elements, such as antibodies, asdescribed herein. A plurality of activatable elements may be examined.Single cells may be placed into predefined classes, and the status ofthe individual determined based on the classes into which cells arecategorized. In some embodiments, a quantitative analysis of the numberof cells in one or more classes is performed to determine the status ofthe individual. In some embodiments, the status to be determinedincludes no return of malignancy, return of malignancy, appearance of anew pathology, e.g., malignancy, which may be a result of treatment, ora combination (e.g., return of malignancy and appearance of a newpathology). Diagnosis, prognosis, and/or a course of treatment may alsobe determined based on the analysis of the classes of cells. See Haskellet al, Cancer Treatment, 5^(th) Ed., W.B. Saunders and Co., 2001.

In some embodiments, the invention provides a method of detecting theminimal residual status of disease in an individual who has receivedtreatment comprising subjecting a plurality of cells in a sample from anindividual to a modulator; determining the activation levels of aplurality of intracellular activatable elements in single cells inresponse to the modulator by a process comprising the binding of aplurality of binding elements which are specific to a particularactivation state of a particular activatable element, wherein the singlecells are placed into one or more classes based on said response to saidmodulator or modulators; determining the presence or absence of saiddisease-associated cells based on the response, wherein determining thepresence or absence of the disease-associated cells comprisesquantitative analysis of the one or more classes; and determining theminimal residual status of a disease, wherein the minimal residualstatus is based on the presence or absence of a small number of thedisease-associated cells.

In some embodiments, diagnosis, prognosis and/or selection of treatmentcourse of a disease comprises tracking the emergence and disappearanceof rare cell populations.

In some embodiments, the determination of status is the presence ofresidual malignant cells, even when there are so few cancer cellspresent (e.g., even one cancer cell) that they cannot be found byroutine diagnostic modalities. The detection of residual malignant cellsindicates that treatment is incomplete. The methods of the invention canthus distinguish between individuals who need additional intensive andpotentially more toxic therapy from those individuals who do not.

In some embodiments, the determination of status comprises the presenceand characteristics of cancer stem cells, which are a very low minorityof the whole tumor cells. Cancer stem cells frequently responddifferently to therapeutic agents than do other tumor cells.Understanding these differences may be important in increasing the curerates for cancer. Cancer stem cell characteristics that may bedetermined include chemotherapy or biological therapy target expressionand response to therapy.

In some embodiments, the determination of status comprises the detectionand functional characterization of immune cells specifically related tothe pathogenesis of autoimmune diseases. Specific immune cells can bemonitored over time while they are under therapeutic pressure either invitro or in vivo to provide information to guide patient management.

Numerous immunologic, proliferative and malignant diseases and disordersare especially amenable to the methods described herein. Immunologicdiseases and disorders include allergic diseases and disorders,disorders of immune function, and autoimmune diseases and conditions.Allergic diseases and disorders include but are not limited to allergicrhinitis, allergic conjunctivitis, allergic asthma, atopic eczema,atopic dermatitis, and food allergy. Immunodeficiencies include but arenot limited to severe combined immunodeficiency (SCID),hypereosinophilic syndrome, chronic granulomatous disease, leukocyteadhesion deficiency I and II, hyper IgE syndrome, Chediak Higashi,neutrophilias, neutropenias, aplasias, Agammaglobulinemia, hyper-IgMsyndromes, DiGeorge/Velocardial-facial syndromes and Interferongamma-TH1 pathway defects. Autoimmune and immune dysregulation disordersinclude but are not limited to rheumatoid arthritis, diabetes, systemiclupus erythematosus, Graves' disease, Graves opthalmopathy, Crohn'sdisease, multiple sclerosis, psoriasis, systemic sclerosis, goiter andstruma lymphomatosa (Hashimoto's thyroiditis, lymphadenoid goiter),alopecia aerata, autoimmune myocarditis, lichen sclerosis, autoimmuneuveitis, Addison's disease, atrophic gastritis, myasthenia gravis,idiopathic thrombocytopenic purpura, hemolytic anemia, primary biliarycirrhosis, Wegener's granulomatosis, polyarteritis nodosa, andinflammatory bowel disease, allograft rejection and tissue destructivefrom allergic reactions to infectious microorganisms or to environmentalantigens.

Proliferative diseases and disorders that may be evaluated by themethods of the invention include, but are not limited to,hemangiomatosis in newborns; secondary progressive multiple sclerosis;chronic progressive myelodegenerative disease; neurofibromatosis;ganglioneuromatosis; keloid formation; Paget's Disease of the bone;fibrocystic disease (e.g., of the breast or uterus); sarcoidosis;Peronies and Duputren's fibrosis, cirrhosis, atherosclerosis andvascular restenosis.

Malignant diseases and disorders that may be evaluated by the methods ofthe invention include both hematologic malignancies and solid tumors.

Hematologic malignancies are especially amenable to the methods of theinvention when the sample is a blood sample, because such malignanciesinvolve changes in blood-borne cells. Such malignancies includenon-Hodgkin's lymphoma, Hodgkin's lymphoma, non-B cell lymphomas, andother lymphomas, acute or chronic leukemias, polycythemias,thrombocythemias, multiple myeloma, myelodysplastic disorders,myeloproliferative disorders, myelofibroses, atypical immunelymphoproliferations and plasma cell disorders.

Plasma cell disorders that may be evaluated by the methods of theinvention include multiple myeloma, amyloidosis and Waldenstrom'smacroglobulinemia.

Leukemias that may be evaluated by the invention include both myeloidand lymphoid leukemias. Myeloid leukemias include AML, CML, and juvenilemyelomonocytic leukemia (JMML). Lymphoid leukemias include non-B cellacute lymphocytic leukemia (T-ALL), and B cell acute lymphoblasticleukemia (including pre-B cell) and chronic lymphocytic leukemia (CLL).Other hematologic diseases and disorders that may be evaluated by themethods of this invention include myeloid disorders such asmyelodysplastic disorders, myeloproliferative disorders, myelofibroses,polycythemias, and thrombocythemias and others such as B cellimmunoproliferations (post transplant lymphoproliferation disorder(PTLD) and non-B atypical immune lymphoproliferations. See Haskell etal, Cancer Treatment, 5^(th) Ed., W.B. Saunders and Co., 2001.

In some embodiments of the invention, the hematologic disease that isevaluated by the methods of the invention is CLL. Thus, in someembodiments the invention provides tracking of the disease courseincluding the emergence and disappearance of rare cell populations,allowing for methods for diagnosing CLL, determining a method oftreatment for CLL, determining a prognosis for CLL, or determiningresponse to treatment for CLL in an individual, using the methodsdescribed herein. In some embodiments, the individual has beenpreviously diagnosed with CLL and is undergoing or has undergonetreatment for CLL. One or more blood samples are taken from theindividual; in some embodiments a series of blood samples are taken fromthe individual over time. The samples may be taken before, during, orafter treatment, or some combination thereof. In some embodiments, thesamples are taken before, during, and after treatment. Additionalsamples or other diagnostic markers, as are known in the art, may alsobe used in addition to the blood samples to determine the status of theindividual; e.g., bone marrow samples may be taken, and/or blood cellsmay examined for well-established markers of CLL, such as surfaceantigen markers, e.g., coexpression of CD5 with CD19 and CD23 or CD5with CD20 and CD23 and dim surface immunoglobulin expression. In someembodiments, the samples or portions of the samples are treated with amodulator, and the state of single cells is determined, from which adetermination is made as to the status of the CLL in the individual. Insome embodiments, the state of single cells is the activation level ofone or more activatable elements, e.g., proteins such asphosphoproteins, in the cells. Quantitative analysis, as describedherein, is performed, in order to determine the status of the CLL in theindividual. In some embodiments, a treatment decision is made based atleast in part on the determination of the status of CLL using themethods described herein; such treatment decision may include notreatment, treatment with a previously-used treatment, modification oftreatment, or use of a new treatment.

In some embodiments, the number of cells associated with CLL may bedeterminative when the number of cells is fewer than 10⁻³ to 10⁻⁴ cells.For example, the presence of 1×10⁻³, 2×10⁻³, 3×10⁻³, 4×10⁻³, 5×10⁻³,6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³, 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁴,6×10⁻⁴, 7×10⁴, 8×10⁻⁴, or 9×10⁻⁴ cells associated with CLL may beindicative of an individual's status. In some embodiments, the number ofcells associated with CLL may be determinative when the number of cellsis higher than 10⁻² to 10⁻⁴ cells. For example, the presence of 1×10⁻²,2×10⁻², 3×10⁻², 4×10⁻², 5×10⁻², 6×10⁻², 7×10⁻², 8×10⁻², 9×10⁻², 1×10⁻³,2×10⁻³, 3×10⁻³, 4×10⁻³, 5×10⁻³, 6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³ or1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁴, 6×10⁻⁴, 7×10⁴, 8×10⁻⁴, or 9×10⁻⁴cells associated with CLL may be indicative of an individual's status.In some embodiments, the number of cells associated with CLL may bedeterminative when it is correlated with a predetermined clinicalparameter. For example in determining the probability of relapse in CLLpatients, patients with specific cell surface proteins or older thancertain age would have relapses if they have number of cells associatedwith CLL higher than for example 10⁻², whereas patients with differentcell surface proteins or younger than certain age would have relapses ifthey have number of cells associated with CLL higher than for example10⁻⁴.

In some embodiments of the invention, the hematologic disease that isevaluated by the methods of the invention is AML. Thus, in someembodiments, the invention provides methods for diagnosing AML,determining a method of treatment for AML, determining a prognosis forAML, or determining response to treatment for AML in an individual,using the methods described herein. In some embodiments, the individualhas been previously diagnosed with AML and is undergoing or hasundergone treatment for AML. One or more blood samples are taken fromthe individual; in some embodiments a series of blood samples are takenfrom the individual over time. The samples may be taken before, during,or after treatment, or some combination thereof. In some embodiments,the samples are taken before, during, and after treatment. Additionalsamples or other diagnostic markers, as are known in the art, may alsobe used in addition to the blood samples to determine the status of theindividual; e.g., bone marrow samples may be taken, and/or blood cellsmay examined for well-established markers of AML including, but are notlimited to, fetal liver tyrosine kinase/internal tandem duplication(FLT3/ITD), NPM1, ERG, KIT, thymidine-kinase expression levels,β2-microglobulin expression, the presence of MDR1 phenotype, orcytogenetic analysis to examine for the presence of abnormal karyotypes.In some embodiments diagnosis, prognosis, or method of treatment furtherrelies on medical history and physical examination including, but notlimited to past bone marrow or peripheral blood stem celltransplantation; total body irradiation with concurrent bone marrow orstem cell transplantation or any combination thereof. In someembodiments, the samples or portions of the samples are treated with amodulator, and the activation level of single cells is determined, fromwhich a determination is made as to the status of the AML in theindividual. In some embodiments, the activation level of single cells isthe activation level of one or more activatable elements, e.g., proteinssuch as phosphoproteins, in the cells. Quantitative analysis, asdescribed herein, is performed, in order to determine the status of theAML in the individual. In some embodiments, a treatment decision is madebased at least in part on the determination of the status of AML usingthe methods described herein; such treatment decision may include notreatment, treatment with a previously-used treatment, modification oftreatment, or use of a new treatment.

In some embodiments, the number of cells associated with AML may bedeterminative when the number of cells is fewer than 10⁻³ to 10⁻⁴ cells.For example, the presence of 1×10⁻³, 2×10⁻³, 3×10⁻³, 4×10⁻³, 5×10⁻³,6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³, 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁴,6×10⁻⁴, 7×10⁴, 8×10⁻⁴, or 9×10⁻⁴ cells associated with AML may beindicative of an individual's status. In some embodiments, the number ofcells associated with AML may be determinative when the number of cellsis higher than 10⁻² to 10⁻⁴ cells. For example, the presence of 1×10⁻²,2×10⁻², 3×10⁻², 4×10⁻², 5×10⁻², 6×10⁻², 7×10⁻², 8×10⁻², 9×10⁻², 1×10⁻³,2×10⁻³, 3×10⁻³, 4×10⁻³, 5×10⁻³, 6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³ or1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁻⁴, 6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or9×10⁻⁴ cells associated with AML may be indicative of an individual'sstatus. In some embodiments, the number of cells associated with AML maybe determinative when it is correlated with a predetermined clinicalparameter. For example in determining the probability of relapse in AMLpatients, patients that have a favorable cytogenetic subtype would haverelapses if they have number of cells associated with AML higher thanfor example 10⁻², whereas patients with adverse cytogenetic subtypes(e.g. (15;17) PML-RARA, t(8;21) AML1-RUNX1T1 (AML-ETO), inv(16)) wouldhave relapses if they have number of cells associated with AML higherthan for example 10⁻⁴.

In some embodiments of the invention, the hematologic disease that isevaluated by the methods of the invention is ALL. Thus, in someembodiments the invention provides methods for diagnosing ALL,determining a method of treatment for ALL, determining a prognosis forALL, or determining response to treatment for ALL in an individual,using the methods described herein. In some embodiments, the individualhas been previously diagnosed with ALL and is undergoing or hasundergone treatment for ALL. One or more blood samples are taken fromthe individual; in some embodiments a series of blood samples are takenfrom the individual over time. The samples may be taken before, during,or after treatment, or some combination thereof. In some embodiments,the samples are taken before, during, and after treatment. Additionalsamples or other diagnostic markers, as are known in the art, may alsobe used in addition to the blood samples to determine the status of theindividual; e.g., bone marrow samples may be taken, and/or blood cellsmay examined for well-established markers of ALL. In some embodiments,the samples or portions of the samples are treated with a modulator, andthe activation level of single cells is determined, from which adetermination is made as to the status of the ALL in the individual. Insome embodiments, the activation level of single cells is the activationlevel of one or more activatable elements, e.g., proteins such asphosphoproteins, in the cells. Quantitative analysis, as describedherein, is performed, in order to determine the status of the ALL in theindividual. In some embodiments, a treatment decision is made based atleast in part on the determination of the status of ALL using themethods described herein; such treatment decision may include notreatment, treatment with a previously-used treatment, modification oftreatment, or use of a new treatment.

In some embodiments, the number of cells associated with ALL may bedeterminative when the number of cells is fewer than 10⁻³ to 10⁻⁴ cells.For example, the presence of 1×10⁻³, 2×10⁻³, 3×10⁻³, 4×10⁻³, 5×10⁻³,6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³, 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁴,6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or 9×10⁻⁴ cells associated with ALL may beindicative of an individual's status. In some embodiments, the number ofcells associated with ALL may be determinative when the number of cellsis higher than 10⁻² to 10⁻⁴ cells. For example, the presence of 1×10⁻²,2×10⁻², 3×10⁻², 4×10⁻², 5×10⁻², 6×10⁻², 7×10⁻², 8×10⁻², 9×10⁻², 1×10⁻³,2×10⁻³, 3×10⁻³, 4×10⁻³, 5×10⁻³, 6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³ or1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁻⁴, 6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or9×10⁻⁴ cells associated with ALL may be indicative of an individual'sstatus. In some embodiments, the number of cells associated with ALL maybe determinative when it is correlated with a predetermined clinicalparameter. For example in determining the probability of relapse in ALLpatients, patients that have a favorable cytogenetic subtype would haverelapses if they have number of cells associated with ALL higher thanfor example 10⁻², whereas patients with adverse cytogenetic subtype(e.g., t(9;22) BCR-ABL, t(12;21) ETV6-RUNX1 (TEL-AML1)) would haverelapses if they have number of cells associated with ALL higher thanfor example 10⁻⁴.

In some embodiments of the invention, the hematologic disease that isevaluated by the methods of the invention is CML. Thus, in someembodiments the invention provides methods for diagnosing CML,determining a method of treatment for CML, determining a prognosis forCML, or determining response to treatment for CML in an individual,using the methods described herein. In some embodiments, the individualhas been previously diagnosed with CML and is undergoing or hasundergone treatment for CML. One or more blood samples are taken fromthe individual; in some embodiments a series of blood samples are takenfrom the individual over time. The samples may be taken before, during,or after treatment, or some combination thereof. In some embodiments,the samples are taken before, during, and after treatment. Additionalsamples or other diagnostic markers, as are known in the art, may alsobe used in addition to the blood samples to determine the status of theindividual; e.g., bone marrow samples may be taken, and/or blood cellsmay examined for well-established markers of CML. In some embodiments,the samples or portions of the samples are treated with a modulator, andthe state of single cells is determined, from which a determination ismade as to the status of the CML in the individual. In some embodiments,the state of single cells is the activation level of one or moreactivatable elements, e.g., proteins such as phosphoproteins, in thecells. Quantitative analysis, as described herein, is performed, inorder to determine the status of the CML in the individual. In someembodiments, a treatment decision is made based at least in part on thedetermination of the status of CML using the methods described herein;such treatment decision may include no treatment, treatment with apreviously-used treatment, modification of treatment, or use of a newtreatment.

In some embodiments, the number of cells associated with CML may bedeterminative when the number of cells is fewer than 10⁻³ to 10⁻⁴ cells.For example, the presence of 1×10⁻³, 2×10⁻³, 3×10⁻³, 4×10⁻³, 5×10⁻³,6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³, 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁻⁴,6×10⁻⁴, 7×10⁴, 8×10⁻⁴, or 9×10⁻⁴ cells associated with CML may beindicative of an individual's status. In some embodiments, the number ofcells associated with CML may be determinative when the number of cellsis higher than 10⁻² to 10⁻⁴ cells. For example, the presence of 1×10⁻²,2×10⁻², 3×10⁻², 4×10⁻², 5×10⁻², 6×10⁻², 7×10⁻², 8×10⁻², 9×10⁻², 1×10⁻³,2×10⁻³, 3×10⁻³, 4×10⁻³, 5×10⁻³, 6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³ or1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁴, 6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or 9×10⁻⁴cells associated with CML may be indicative of an individual's status.In some embodiments, the number of cells associated with CML may bedeterminative when it is correlated with a predetermined clinicalparameter. For example in determining the probability of relapse in CMLpatients, patients that have a favorable cytogenetic subtype would haverelapses if they have number of cells associated with CML higher thanfor example 10⁻², whereas patients with adverse cytogenetic subtype(e.g., t(9;22) BCR-ABL) would have relapses if they have number of cellsassociated with CML higher than for example 10⁻⁴.

In some embodiments of the invention, the hematologic disease that isevaluated by the methods of the invention is follicular lymphoma. Thus,in some embodiments the invention provides methods for diagnosingfollicular lymphoma, determining a method of treatment for follicularlymphoma, determining a prognosis for follicular lymphoma, ordetermining response to treatment for follicular lymphoma in anindividual, using the methods described herein. In some embodiments, theindividual has been previously diagnosed with follicular lymphoma and isundergoing or has undergone treatment for follicular lymphoma. One ormore blood samples are taken from the individual; in some embodiments aseries of blood samples are taken from the individual over time. Thesamples may be taken before, during, or after treatment, or somecombination thereof. In some embodiments, the samples are taken before,during, and after treatment. Additional samples or other diagnosticmarkers, as are known in the art, may also be used in addition to theblood samples to determine the status of the individual; e.g., bonemarrow samples may be taken, and/or blood cells may examined forwell-established markers of follicular lymphoma. In some embodiments,the samples or portions of the samples are treated with a modulator, andthe state of single cells is determined, from which a determination ismade as to the status of the follicular lymphoma in the individual. Insome embodiments, the activation level of single cells is the activationlevel of one or more activatable elements, e.g., proteins such asphosphoproteins, in the cells. Quantitative analysis, as describedherein, is performed, in order to determine the status of the follicularlymphoma in the individual. In some embodiments, a treatment decision ismade based at least in part on the determination of the status offollicular lymphoma using the methods described herein; such treatmentdecision may include no treatment, treatment with a previously-usedtreatment, modification of treatment, or use of a new treatment.

In some embodiments, the number of cells associated with follicularlymphoma may be determinative when the number of cells is fewer than10⁻³ to 10⁻⁴ cells. For example, the presence of 1×10⁻³, 2×10⁻³, 3×10⁻³,4×10⁻³, 5×10⁻³, 6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³, 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴,4×10⁻⁴, 5×10⁴, 6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or 9×10⁻⁴ cells associated withfollicular lymphoma may be indicative of an individual's status. In someembodiments, the number of cells associated with follicular lymphoma maybe determinative when the number of cells is higher than 10⁻² to 10⁻⁴cells. For example, the presence of 1×10⁻², 2×10⁻², 3×10⁻², 4×10⁻²,5×10⁻², 6×10⁻², 7×10⁻², 8×10⁻², 9×10⁻², 1×10⁻³, 2×10⁻³, 3×10⁻³, 4×10⁻³,5×10⁻³, 6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³ or 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴,4×10⁻⁴, 5×10⁻⁴, 6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or 9×10⁻⁴ cells associated withfollicular lymphoma may be indicative of an individual's status. In someembodiments, the number of cells associated with follicular lymphoma maybe determinative when it is correlated with a predetermined clinicalparameter. For example in determining the probability of relapse infollicular lymphoma patients, patients that have a favorable cytogeneticsubtype would have relapses if they have number of cells associated withfollicular lymphoma higher than for example 10⁻², whereas patients withadverse cytogenetic subtype (e.g., t(14;18) IgH/BCL2) would haverelapses if they have number of cells associated with follicularlymphoma higher than for example 10⁻⁴.

In some embodiments of the invention, the hematologic disease that isevaluated by the methods of the invention is mantle cell lymphoma. Thus,in some embodiments the invention provides methods for diagnosing mantlecell lymphoma, determining a method of treatment for mantle celllymphoma, determining a prognosis for mantle cell lymphoma, ordetermining response to treatment for mantle cell lymphoma in anindividual, using the methods described herein. In some embodiments, theindividual has been previously diagnosed with mantle cell lymphoma andis undergoing or has undergone treatment for mantle cell lymphoma. Oneor more blood samples are taken from the individual; in some embodimentsa series of blood samples are taken from the individual over time. Thesamples may be taken before, during, or after treatment, or somecombination thereof. In some embodiments, the samples are taken before,during, and after treatment. Additional samples or other diagnosticmarkers, as are known in the art, may also be used in addition to theblood samples to determine the status of the individual; e.g., bonemarrow samples may be taken, and/or blood cells may examined forwell-established markers of mantle cell lymphoma. In some embodiments,the samples or portions of the samples are treated with a modulator, andthe state of single cells is determined, from which a determination ismade as to the status of the mantle cell lymphoma in the individual. Insome embodiments, the state of single cells is the activation level ofone or more activatable elements, e.g., proteins such asphosphoproteins, in the cells. Quantitative analysis, as describedherein, is performed, in order to determine the status of the mantlecell lymphoma in the individual. In some embodiments, a treatmentdecision is made based at least in part on the determination of thestatus of mantle cell lymphoma using the methods described herein; suchtreatment decision may include no treatment, treatment with apreviously-used treatment, modification of treatment, or use of a newtreatment.

In some embodiments, the number of cells associated with mantle celllymphoma may be determinative when the number of cells is fewer than10⁻³ to 10⁻⁴ cells. For example, the presence of 1×10⁻³, 2×10⁻³, 3×10⁻³,4×10⁻³, 5×10⁻³, 6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³, 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴,4×10⁻⁴, 5×10⁻⁴, 6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or 9×10⁻⁴ cells associated withmantle cell lymphoma may be indicative of an individual's status. Insome embodiments, the number of cells associated with mantle celllymphoma may be determinative when the number of cells is higher than10⁻² to 10⁻⁴ cells. For example, the presence of 1×10⁻², 2×10⁻², 3×10⁻²,4×10⁻², 5×10⁻², 6×10⁻², 7×10⁻², 8×10⁻², 9×10⁻², 1×10⁻³, 2×10⁻³, 3×10⁻³,4×10⁻³, 5×10⁻³, 6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³ or 1×10⁻⁴, 2×10⁻⁴,3×10⁻⁴, 4×10⁻⁴, 5×10⁻⁴, 6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or 9×10⁻⁴ cellsassociated with mantle cell lymphoma may be indicative of anindividual's status. In some embodiments, the number of cells associatedwith mantle cell lymphoma may be determinative when it is correlatedwith a predetermined clinical parameter. For example in determining theprobability of relapse in mantle cell lymphoma patients, patients thathave a favorable cytogenetic subtype would have relapses if they havenumber of cells associated with mantle cell lymphoma higher than forexample 10⁻², whereas patients with adverse cytogenetic subtype (e.g.,t(11;14) IgH/CCND1 (IgH/BCL1)) would have relapses if they have numberof cells associated with mantle cell lymphoma higher than for example10⁻⁴.

In some embodiments of the invention, the hematologic disease that isevaluated by the methods of the invention is multiple myeloma. Thus, insome embodiments the invention provides methods for diagnosing multiplemyeloma, determining a method of treatment for multiple myeloma,determining a prognosis for multiple myeloma, or determining response totreatment for multiple myeloma in an individual, using the methodsdescribed herein. In some embodiments, the individual has beenpreviously diagnosed with multiple myeloma and is undergoing or hasundergone treatment for multiple myeloma. One or more blood samples aretaken from the individual; in some embodiments a series of blood samplesare taken from the individual over time. The samples may be takenbefore, during, or after treatment, or some combination thereof. In someembodiments, the samples are taken before, during, and after treatment.Additional samples or other diagnostic markers, as are known in the art,may also be used in addition to the blood samples to determine thestatus of the individual; e.g., bone marrow samples may be taken, and/orblood cells may examined for well-established markers of multiplemyeloma. In some embodiments, the samples or portions of the samples aretreated with a modulator, and the state of single cells is determined,from which a determination is made as to the status of the multiplemyeloma in the individual. In some embodiments, the activation level ofsingle cells is the activation level of one or more activatableelements, e.g., proteins such as phosphoproteins, in the cells.Quantitative analysis, as described herein, is performed, in order todetermine the status of the multiple myeloma in the individual. In someembodiments, a treatment decision is made based at least in part on thedetermination of the status of multiple myeloma using the methodsdescribed herein; such treatment decision may include no treatment,treatment with a previously-used treatment, modification of treatment,or use of a new treatment.

In some embodiments, the number of cells associated with multiplemyeloma may be determinative when the number of cells is fewer than 10⁻³to 10⁻⁴ cells. For example, the presence of 1×10⁻³, 2×10⁻³, 3×10⁻³,4×10⁻³, 5×10⁻³, 6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³, 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴,4×10⁻⁴, 5×10⁻⁴, 6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or 9×10⁻⁴ cells associated withmultiple myeloma may be indicative of an individual's status. In someembodiments, the number of cells associated with multiple myeloma may bedeterminative when the number of cells is higher than 10⁻² to 10⁻⁴cells. For example, the presence of 1×10⁻², 2×10⁻², 3×10⁻², 4×10⁻²,5×10⁻², 6×10⁻², 7×10⁻², 8×10⁻², 9×10⁻², 1×10⁻³, 2×10⁻³, 3×10⁻³, 4×10⁻³,5×10⁻³, 6×10⁻³, 7×10⁻³, 8×10⁻³, 9×10⁻³ or 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴,4×10⁻⁴, 5×10⁻⁴, 6×10⁻⁴, 7×10⁻⁴, 8×10⁻⁴, or 9×10⁻⁴ cells associated withmultiple myeloma may be indicative of an individual's status. In someembodiments, the number of cells associated with multiple myeloma may bedeterminative when it is correlated with a predetermined clinicalparameter. For example in determining the probability of relapse inmultiple myeloma patients, patients with specific cell surface proteinsor having high levels of somatic hypermutations would have relapses ifthey have number of cells associated with multiple myeloma higher thanfor example 10⁻², whereas patients with different cell surface proteinsor low levels of somatic hypermutations would have relapses if they havenumber of cells associated with multiple myeloma higher than for example10⁻⁴.

In some embodiments of the invention, disease that is evaluated by themethods of the invention is a solid tumor. Thus, in some embodiments theinvention provides methods for diagnosing solid tumors, determining amethod of treatment for solid tumors, determining the prognosis of apatient with solid tumors, or determining response to treatment of solidtumors in an individual, using the methods described herein. In someembodiments, the individual has been previously diagnosed with a solidtumor and has undergone treatment for a solid tumor. One or more samplesare taken from the individual; in some embodiments a series of samplesare taken from the individual over time. The samples may be takenbefore, during, or after treatment, or some combination thereof. In someembodiments, the samples are taken before, during, and after treatment.Samples may be blood samples, lymph node samples, other appropriatesamples (dependent on the solid tumor type), or a combination of sampletypes. Additional samples or other diagnostic markers, as are known inthe art, may also be used in addition to the samples analyzed for thestate of individual cells. In some embodiments, the samples or portionsof the samples are treated with a modulator, and the state of singlecells is determined, from which a determination is made as to the statusof the solid tumor in the individual. In some embodiments, the state ofsingle cells is the activation level of one or more activatableelements, e.g., proteins such as phosphoproteins, in the cells.Quantitative analysis, as described herein, is performed, in order todetermine the status of the solid tumor in the individual. In someembodiments, a treatment decision is made based at least in part on thedetermination of the status of the solid tumor using the methodsdescribed herein; such treatment decision may include no treatment,treatment with a previously-used treatment, modification of treatment,or use of a new treatment. The solid tumor may be any solid tumoramenable to sampling for direct or indirect analysis; solid tumorsinclude but are not limited to head and neck cancer including brain,thyroid cancer, breast cancer, lung cancer, mesothelioma, germ celltumors, ovarian cancer, liver cancer, gastric carcinoma, colon cancer,prostate cancer, pancreatic cancer, melanoma, bladder cancer, renalcancer, prostate cancer, testicular cancer, cervical cancer, endometrialcancer, myosarcoma, leiomyosarcoma and other soft tissue sarcomas,osteosarcoma, Ewing's sarcoma, retinoblastoma, rhabdomyosarcoma, Wilm'stumor, and neuroblastoma.

Once the status of an individual (e.g., health status) is determined, anappropriate therapeutic action can be taken. The appropriate therapeuticaction can take many forms: in the case of cancer, surgery,transplantation, or the administration of a physical, chemical, orbiological agent, or combinations thereof. For some individuals, theappropriate action is to initiate a new therapy either in addition tothe current therapy or in place of it. For others, a new therapy is notindicated, but instead, the existing therapy should be continued,perhaps in a modified form such as escalating the dosage of amedication. In still other individuals, the existing course of therapyshould be shortened, while in others it should be lengthened. In someindividuals, the appropriate action is to stop the existing therapywithout initiating another form of therapy. In some individuals, theappropriate action is to start supportive care.

In some instances, the appropriate therapy is surgery, of which,numerous forms are known including excisional surgery, cryosurgery, orlaser surgery. Surgery can be performed for preventative, curative, orpalliative goals. If a predefined class is associated with an elevatedrisk of developing an organ or tissue specific disease such as breast,colon, or ovarian cancer, prophylactic surgery can be performed toremove the organ or tissue.

In other instances, the appropriate therapy is transplantation.Transplantation includes the transplantation of whole or partial organs,tissues or stem cells from allogenic, autologous, syngenic or xenogenicorigin. Stem cells can be derived from peripheral blood, umbilical cord,embryos, bone marrow or other organs and tissue.

In some instances, the appropriate therapy is radiation also known asradiotherapy. Radiation is either electromagnetic or particulate and canbe administered by external beam, brachytherapy, or by theadministration of radioactive substances including elements,nucleotides, drugs, radiolabeled peptides or radiolabeled antibodies.

In still other instances, the appropriate therapy is the administrationof a chemical agent or drug. Such agents comprise a diverse group andcan be categorized in numerous ways including by function, chemicalstructure, or cellular or molecular target.

In one embodiment, the appropriate therapy is the administration of achemical agent that is a chemotherapy agent used to treat malignancies.Chemotherapeutic agents include, but are not limited to, alkylatingagents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including synthetic analogue topotecan);bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesinand bizelesin synthetic analogues); cryptophycins (particularlycryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (includingthe synthetic analogues, KW-2189 and CBI-TMI); eleutherobin;pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such aschlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosoureas such as carmustine, chlorozotocin, foremustine, lomustine,nimustine, ranimustine; antibiotics such as the enediyne antibiotics(e.g. calicheamicin); dynemicin, including dynemicin A; bisphosphonates,such as clodronate; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibioticchromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubincin (Adramycin™) (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as demopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogues such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replinisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane;rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethane; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiopeta; taxoids,e.g. paclitaxel (TAXOL™) and docetaxel (TAXOTERE™); chlorambucil;gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitroxantrone; vancristine;vinorelbine (Navelbine™); novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeoloda; ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid; capecitabine; and pharmaceutically acceptable salts,acids or derivatives of any of the above. See Haskell et al, CancerTreatment, 5^(th) Ed., W.B. Saunders and Co., 2001.

Also included in the definition of “chemotherapeutic agent” areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen, raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston™); inhibitors of the enzyme aromatase, whichregulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, megestrol acetate(Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™),letrozole (Femara™), and anastrozole (Arimidex™); and anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

In another embodiment, the appropriate therapy is the administration ofa chemical agent that is a targeted therapy drug. For the treatment ofmalignancies, targeted therapeutics include, but are not limited toimatinib mesylate (Gleevec™, also known as STI-571; gefitinib (Iressa™,also known as ZD1839), erlotinib; bortezomib (Velcade™); and oblimersen(Genasense™).

In a further embodiment, the appropriate therapy is the administrationof a biological agent comprising native and engineered antibodiesincluding antibodies conjugated to drugs and toxins, antisenseoligonucleotides, RNA interference oligonucleotides, peptides, hormones,cytokines, biological response modifiers, vaccines, growth factors,natural products, and ex-vivo expanded tumor-infiltrating lymphocytes.

Biological agents comprise native or engineered antibodies, includingantibodies conjugated to drugs and toxins, antisense oligonucleotides,RNA interference oligonucleotides, peptides, hormones, cytokines,biological response modifiers, vaccines, growth factors, naturalproducts, and ex-vivo expanded tumor-infiltrating lymphocytes.

An example of an antibody useful for treating breast cancer istrastuzumab. This antibody recognizes a member of the human epidermalgrowth factor receptor (HER) family of transmembrane tyrosine kinasesHER2/neu (ErbB2).

The determination of the appropriate therapy for an individual may alsorequire assessing one or more other individual characteristics includingphysical characteristics, clinical status, previous treatmentcharacteristics, and biochemical/molecular markers. Individualcharacteristics may further comprise patient's past medical history,family medical history, patient's social history, as well as any currentmedical history termed “review of systems.”

Physical characteristics include an individual's gender; current age;age at the time of disease presentation; age at the time of treatment.Clinical status includes clinical stage of disease, performance status,blood cell count; bone marrow reserves. Factors from previous treatmentsthat can be considered include type of previous therapies, number ofprevious therapies, response to previous therapy or therapies and timefrom last treatment. Biochemical and molecular markers include thosethat serve to define known patient response or outcome to a giventherapy. Also included are markers of drug metabolism phenotypes such ascytochrome p450 isoforms.

Determination of response to treatment may comprise the assessment ofother factors such as whether there was complete or partial resolutionof symptoms, normalization of clinical parameters such as cell counts,or blood chemistry, a reduction in pain or other subjectivemeasurements, or a reduction in pain medication, transfusions, oxygen orother supportive requirements.

EXAMPLES Example 1 Identification of Subpopulations of Bone Marrow Cellsfrom Normal Individuals and MDS Patients Objectives and Study Design:

The objectives of the study were to determine whether cyropreservedsamples can be used to characterize MDS and to determine whether adistinct subpopulation of nucleated red blood cells (nRBCs) can beidentified in MDS patients. This study was also to design a modulatorand staining panel for characterizing responses of MDS patient cellpopulations including myeloblasts, monocytes, lymphocytes and nRBCs atdifferent developmental stages in response to different stimuliincluding EPO, IFNγ, FLT3, SCF, and PMA. The modulator and stainingpanel is shown in Table 1 below.

TABLE 1 Priority Modulator Stain 1 Surface Erythroid Precursor: CD71,Phenotype CD235ab 2 Surface Stem Cell: CD117, CD38 Phenotype 3 SurfaceCD45 Isoforms: CD45RA, Phenotype CD45RO, CD45RB 4 Surface Autoimmune:CD3, CD4, CD8 Phenotype 5 Unstim STAT1/3/5 6 EPO STAT1/3/5 7 EPO + G-CSFSTAT1/3/5 8 G-CSF STAT1/3/5 9 IL-3 STAT1/3/5 10 IFN-g STAT1/3/5 11Unstim Erk, S6, Akt 12 SCF Erk, S6, Akt 13 FLT3L Erk, S6, Akt 14 PMAErk, S6, Akt 15 SDF-1a Erk, S6, Akt 16 Unstim Chk2, cleaved PARP 17Etoposide Chk2, cleaved PARP 18 Unstim Caspase 8, cleaved PARP 19Etoposide Caspase 8, cleaved PARP 20 Unstim NFkB, p38, Erk 21 LPS NFkB,p38, Erk 22 TNF-a NFkB, p38, Erk 23 EPO STAT1/3/5 24 EPO + G-CSFSTAT1/3/5 25 IL-3 STAT1/3/5 26 IFN-g STAT1/3/5 27 SDF-1a Erk, S6, Akt

In this study, there were five MDS patient samples (01-05) and fivenormal samples (06-10). The clinical information on these 10 samples issummarized in Table 2.

TABLE 2 Classi- Sample fication Age Gender Ethnicity WBC BM Blast Sample01 RA 56 M White 3 1% Sample 02 RAEB 74 F Af. American 8 10% Sample 03RAEB 54 M White 4.7 14% Sample 04 RA 57 M White 3.5 2% Sample 05 RARS 74M White 3.1 0% Sample 06 — 41 F — — — Sample 07 — 23 M — — — Sample 08 —24 M — — — Sample 09 — 45 F — — — Sample 10 — 31 M — — —

Materials and Methods

The present illustrative example represents how to analyze cells in oneembodiment of the present invention. There are several steps in theprocess, such as the step where a modulator is added, the staining stepand the flow cytometry step. The stimulation step of the phospho-flowprocedure can start with vials of frozen cells and end with cells fixedand permeabilized in methanol. Then the cells can be incubated with anantibody directed to a particular protein of interest and then analyzedusing a flow cytometer. A protocol similar to the following is used toanalyze AML cells from patient samples.

The materials used in this invention include thawing medium whichcomprises PBS-CMF+10% FBS+2 mM EDTA; 70 um Cell Strainer (BD); anti-CD45antibody conjugated to Alexa 700 (Invitrogen) used at 1 ul per sample;propidium iodide (PI) solution (Sigma 10 ml, 1 mg/ml) used at 1 ug/ml;RPMI+1% FBS medium; media A comprising RPMI+1% FBS+1× Penn/Strep;Live/Dead Reagent, Amine Aqua (Invitrogen); 2 ml, 96-Deep Well, U-bottompolypropylene plates (Nunc); 300 ul 96-Channel Extended-Length D.A.R.T.tips for Hydra (Matrix); Phosphate Buffered Saline (PBS) (MediaTech);16% Paraformaldehyde (Electron Microscopy Sciences); 100% Methanol (EMD)stored at −20° C.; Transtar 96 dispensing apparatus (Costar); Transtar96 Disposable Cartridges (Costar, Polystyrene, Sterile); Transtarreservoir (Costar); and foil plate sealers.

a. Thawing Cell and Live/Dead Staining:

Frozen cells are thawed in a 37° C. water bath and gently resuspended inthe vial and transferred to the 15 mL conical tube. The 15 mL tube iscentrifuged at 930 RPM (200×g) for 8 minutes at room temperature. Thesupernatant is aspirated and the pellet is gently resuspended in 1 mLmedia A. The cell suspension is filtered through a 70 um cell strainerinto a new 15 mL tube. The cell strainer is rinsed with 1 mL media A andanother 12 ml of media A into the 15 mL tube. The cells are mixed intoan even suspension. A 20 μL aliquot is immediately removed into a96-well plate containing 180 μL PBS+4% FBS+CD45 Alexa 700+PI todetermine cell count and viability post spin. After the determination,the 15 mL tubes are centrifuged at 930 RPM (200×g) for 8 minutes at roomtemperature. The supernatant is aspirated and the cell pellet is gentlyresuspended in 4 mL PBS+4 μL Amine Aqua and incubated for 15 min in a37° C. incubator. 10 mL RPMI+1% FBS is added to the cell suspension andthe tube is inverted to mix the cells. The 15 mL tubes are centrifugedat 930 RPM (200×g) for 8 minutes at room temperature. The cells areresuspended in Media A at the desired cell concentration (1.25×10⁶/mL).For samples with low numbers of cells (<18.5×10⁶), the cells areresuspended in up to 15 mL media. For samples with high numbers of cells(>18.5×10⁶), the volume is raised to 10 mL with media A and the desiredvolume is transferred to a new 15 mL tube, and the cell concentration isadjusted to 1.25×10⁶ cells/ml. 1.6 mL of the above cell suspension(concentration at 1.25×10⁶ cells/ml) is transferred into wells of amulti-well plate. From this plate, 80 ul is dispensed into each well ofa subsequent plate. The plates are covered with a lid (Nunc) and placedin a 37° C. incubator for 2 hours to rest.

b. Addition to a Modulator to the Cells

A concentration for each modulator that is five folds more (5×) than thefinal concentration is prepared using Media A as diluent. 5× stimuli arearrayed into wells of a standard 96 well v-bottom plate that correspondto the wells on the plate with cells to be stimulated.

Preparation of fixative: Stock vial contains 16% paraformaldehyde whichis diluted with PBS to a concentration that is 1.5×. The stock vial isplaced in a 37° C. water bath.

Adding the modulator: The cell plate(s) are taken out of the incubatorand placed in a 37° C. water bath next to the pipette apparatus. Thecell plate is taken from the water bath and gently swirled to resuspendany settled cells. With pipettor, the stimulant is dispensed into thecell plate and vortexed at “7” for 5 seconds. The deep well plate is putback into the water bath.

Adding Fixative: 200 μl of the fixative solution (final concentration at1.6%) is dispensed into wells and then mixed on the titer plate shakeron high for 5 seconds. The plate is covered with foil sealer andincubated in a 37° C. water bath for 10 minutes. The plate is spun for 6minutes at 2000 rpm at room temperature. The cells are aspirated using a96 well plate aspirator (VP Scientific). The plate is vortexed toresuspend cell pellets in the residual volume. The pellet is ensured tobe dispersed before the Methanol step (see cell permeabilization) orclumping will occur.

Cell Permeabilization: Permeability agent, for example methanol, isadded slowly and while the plate is vortexing. To do this, the cellplate is placed on titer plate shaker and made sure it is secure. Theplate is set to shake using the highest setting. A pipetter is used toadd 0.6 mls of 100% methanol to the plate wells. The plate(s) are put onice until this step has been completed for all plates. Plates arecovered with a foil seal using the plate roller to achieve a tight fit.At this stage the plates may be stored at −80° C.

c. Staining Protocol

Reagents for staining include FACS/Stain Buffer-PBS+0.1% Bovine serumalbumen (BSA)+0.05% Sodium Azide; Diluted Bead Mix-1 mL FACS buffer+1drop anti-mouse Ig Beads+1 drop negative control beads. The generalprotocol for staining cells is as follows, although numerous variationson the protocol may be used for staining cells:

Cells are thawed if frozen. Cells are pelleted at 2000 rpm 5 minutes.Supernatant is aspirated with vacuum aspirator. Plate is vortexed on a“plate vortex” for 5-10 seconds. Cells are washed with 1 mL FACS buffer.Repeat the spin, aspirate and vortex steps as above. 50 mL of FACS/stainbuffer with the desired, previously optimized, antibody cocktail isadded to two rows of cells at a time and agitate the plate. The plate iscovered and incubated in a shaker for 30 minutes at room temperature(RT). During this incubation, the compensation plate is prepared. Forthe compensation plate, in a standard 96 well V-bottom plate, 20 mL of“diluted bead mix” is added per well. Each well gets 5 μL of 1fluorophor conjugated control IgG (examples: Alexa488, PE, Pac Blue,Aqua, Alexa647, Alexa700). For the Aqua well, add 200 μL of Aqua−/+cells. Incubate the plate for 10 minutes at RT. Wash by adding 200 μLFACS/stain buffer, centrifuge at 2000 rpm for 5 minutes, and removesupernatant. Repeat the washing step and resuspend the cells/beads in200 μL FACS/stain buffer and transfer to a U-bottom 96 well plate. After30 min, 1 mL FACS/stain buffer is added and the plate is incubated on aplate shaker for 5 minutes at room temperature. Centrifuge, aspirate andvortex cells as described above. 1 mL FACS/stain buffer is added to theplate and the plate is covered and incubated on a plate shaker for 5minutes at room temperature. Repeat the above two steps and resuspendthe cells in 75 μl FACS/stain buffer. The cells are analyzed using aflow cytometer, such as a LSRII (Becton Disckinson). All wells areselected and Loader Settings are described below: Flow Rate: 2 uL/sec;Sample Volume: 40 uL; Mix volume: 40 uL; Mixing Speed: 250 uL/sec; #Mixes: 5; Wash Volume: 800 uL; STANDARD MODE. When a plate hascompleted, a Batch analysis is performed to ensure no clogging.

d. Gating Protocol

Data acquired from the flow cytometer are analyzed with Flowjo software(Treestar, Inc). The Flow cytometry data is first gated on single cells(to exclude doublets) using Forward Scatter Characteristics Area andHeight (FSC-A, FSC-H). Single cells are gated on live cells by excludingdead cells that stain positive with an amine reactive viability dye(Aqua-Invitrogen). Live, single cells are then gated for subpopulationsusing antibodies that recognize surface markers as follows: CD45++,CD33− for lymphocytes, CD45++, CD33++ for monocytes+granulocytes andCD45+, CD33+ for leukemic blasts. Signaling, determined by theantibodies that interact with intracellular signaling molecules, inthese subpopulation gates that select for “lymphs”, “monos+grans, and“blasts” is analyzed.

The data can then be analyzed using various metrics, such as basal levelof a protein or the basal level of phosphorylation in the absence of astimulant, total phosphorylated protein, or fold change (by comparingthe change in phosphorylation in the absence of a stimulant to the levelof phosphorylation seen after treatment with a stimulant), on each ofthe cell populations that are defined by the gates in one or moredimensions. These metrics are then organized in a database tagged by:the Donor ID, plate identification (ID), well ID, gated population,stain, and modulator. These metrics tabulated from the database are thencombined with the clinical data to identify nodes that are correlatedwith a pre-specified clinical variable (for example; response or nonresponse to therapy) of interest.

Results:

Staining of CD45 on myeloblasts, mature monocytes and lymphocytes fromnormal and MDS bone marrow in the presence of PMA shows low variance inCD45 levels among these cell populations, indicating robustness andreproducibility of the CD45 staining (data not shown). For myeblaststimulated with PMA the range for MDS patients was −0.21, −0.31 and therange for normal patients was −0.022, 0.44 and the p value, p-value(Wilcox) and AUC were 0.1584, 0.09524 and 1, respectively. For maturemonocytes stimulated with PMA the range for MDS patients was −0.14,−0.085 and the range for normal patients was −0.26, 0.057 and the pvalue, p-value (wilcox) and AUC were 0.2449, 0.845 and 0.61,respectively. For lymphocytes stimulated with PMA the range for MDSpatients was −0.14, −0.072 and the range for normal patients was −0.059,0.014 and the p value, p-value (wilcox) and AUC were 0.2742, 0.07864 and1, respectively.

Subpopulations of bone marrow mononuclear cells (BMMCs) from normal andMDS patients were gated and identified by flow cytometry. The bonemarrow cells were first gated based on their FSC and SSC profiles, andlive cells were identified as Aqua negative in an Aqua vs. SSC plot.Live cells expressing high levels of CD45 were further plotted and gatedbased on their CD34, CD11b and CD33 expression into CD34+CD11b^(lo)myeloblasts, CD11b+CD33+ mature monocytes, and CD45+SSC^(lo) lymphocytes(FIG. 5). Cells expressing intermediate levels of CD45 were gated asnRBC. nRBCs were further characterized into different developmentalstages based on their CD235ab and CD71 expression profiles (FIG. 5).Subpopulations of lymphocytes, for example, CD3+ T cells were identifiedin normal and MDS bone marrow as CD45+CD3+ (data not shown).Subpopulations of CD3+ T cells, namely, CD4+ and CD8+ T cells in normaland MDS bone marrow were identified based on their surface CD4 and CD8expression (data not shown). FIG. 6 illustrates identification of nRBCsat different developmental stages, i.e. early erythroblasts,normoblasts, and more mature RBCs based on their CD235ab versus CD71expression (see, Hoefsloot L H, Lowenberg B et al. Blood, 1997 Mar. 1;89(5): 1690-700). A comparison of CD235ab versus CD71 expressionprofiles of nRBCs from normal and MDS bone marrow reveals a higherpercentage of CD235+CD71+ normoblasts and a less percentage ofCD235ab-CD71− cells in the MDS bone marrow as compared to the normalbone marrow, suggesting a block of erythroid differentiation in MDS.These results suggest that a rare population of CD235+CD71+ may beinvolved in the pathogenesis of MDS (FIGS. 6 and 7) and can be used forthe diagnosis of MDS.

The results show robustness and reproducibility of staining for rarepopulation of cells. Small numbers of subpopulations of bone marrowcells including subsets of T cells and nRBCs from normal individuals andMDS patients can be identified and used to provide clinical informationthat can be used, for example, in the diagnosis, prognosis, determiningprogression, predicting response to treatment or choosing a treatment.

Example 2 Cellular Responses of Subpopulations of Bone Marrow Cells fromNormal Individuals and MDS Patients

nRBCs (identified in Example 1) from normal individuals and MDSpatients, were stimulated with various stimuli including EPO, IFNγ,FLT3, SCF, PMA, G-CSF and the combinations thereof. The cell stimulationand staining were carried out according to the detailed protocolsdescribed in Example 1.

A variety of fluorochrome-conjugated antibodies that recognize cellsurface and intracellular markers including CD11b, CD33, CD34, CD45,C-casp8, C-PARP, pAkt, pChk2, perk, pNFkb, p-p38, p-S6, pSTAT1, pSTAT3,and pSTAT5 were incubated with the cells. nRBCs from normal individualsand MDS patients were treated with erythropoietin (EPO) and theEPO-mediated Stat5 and Stat1 phosphorylation was assessed by flowcytometry. As shown in FIG. 8, nRBC subpopulation from MDS patientsexhibits Stat5 phosphorylation in response to EPO stimulation. Thisresponse in a small population to EPO stimulation identifies a rare cellpopulation. Interestingly, the shapes of the contour plots, for bothunstimulated and stimulated samples, are different between MDS andNormal patients. FIG. 9 shows Stat5 and Stat1 phosphorylation in rRBCsfrom normal and MDS patients in response to interferon gamma (IFNγ)stimulation. A small nRBC subpopulation from MDS patients exhibits Stat1phosphorylation in response to IFNγ stimulation. These resultsdemonstrate the ability to measure the cellular responses of smallnumbers of cells present in MDS patients. Thus, the methods describedherein can be used to detect a small number of cells, which may berelated to a disease such as cancer and can be used for it diagnosis.

Example 3 Effects of Therapeutics on Healthy Bone Marrow Cells

Live healthy bone marrow mononuclear cells (BMMCs) were contacted withseveral drugs at different concentrations by a 1:3 dilution in themedium, for example, 100 μM, 33.3 μM, 11.1 μM, 3.7 μM, 1.2 μM, 0.4 μM,0.14 μM, 0.046 μM, 0.015 μM, 0.005 M, or 0.0017 μM of 5-Azacytidine(Vidaza), Decitabine (Dacogen), Vorinostat (Zolina) and DMSO. CD45 andCD34 expression was assessed by flow cytometry after 24 hours ofstimulation with each drug. The cell stimulation and staining werecarried out according to the detailed protocols described in Example 1.The CD45 versus CD34 expression profiles of healthy BMMCs exposed to5-Azacytidine (Vidaza), Decitabine (Dacogen), Vorinostat (Zolinza), orDMSO are shown in FIGS. 10-12, respectively. 5-Azacytidine (Vidaza) andDecitabine (Dacogen) are hypomethylating agents. The results shown that5-Azacytidine (Vidaza) results in a dose-dependent loss of a rarepopulation of CD34+ myeloblast cells (FIG. 10). In contrast, Decitabine(Dacogen), a drug in the same molecular class as Vidaza, does not affectthe viability of the rare populationCD34+ myeloblast cells (FIG. 11).Vorinostat (Zolinza), a histone deacetylase (HDAC) inhibitor, showsselective loss of rare population of CD34+ myeloblast cells in adose-dependent fashion (FIG. 12).

The results show that the methods described herein enable themeasurement of drug responses in small populations of cells.

Example 4 CD45RA/RO/RB Expression Profiles of Subpopulations of BoneMarrow Cells from Normal Individuals and MDS Patients

Cells from normal and MDS bone marrows were gated based on their CD45and SSC expression profile as described above. CD45RA, CD45RO and CD45RBexpression on nRBCs was assessed by flow cytometry. CD45RO, CD45RA, andCD45RB are isoforms of CD45. Each CD45 isoforms is distinguished fromone another isoform depending on the type of exon the CD45 has or theexons the CD45 does not have. The CD45RA isoform contains the A exononly and the CD45RB has the B exon only whereas the CD45RO has none ofthe exons: A, B, or C. Altered expression of CD45 isoforms onhematopoietic cells, particularly lymphocytes, has been associated withvarious diseases.

FIGS. 12 and 13 shows CD45RA/RO/RB expression profiles of maturemonocytes, myeloblasts and lymphocytes from normal and MDS bone marrows.Mature monocytes in the bone marrow were gated as CD33^(hi)CD11b^(hi).Myeloblasts were gated as CD34⁺CD11b^(lo), and lymphocytes were gatedbased on their CD45 and SSC expression profiles. CD45RA, CD45RO andCD45RB expressions on monocytes, myeloblasts and lymphocytes wereassessed by flow cytometry. The results show differences in CD45RA/RO/RBlevels between normal individuals and MDS patients among differentsubpopulations of mature monos, blast and lymphocytes. CD45 isoformexpression, thus, identifies unique rare cells populations in MDSpatients

In summary, the study of the present invention suggests thatcryopreserved MDS patient samples can be used to examine myeloblasts,erythroid precursors, monocytes, and lymphocytes in terms of theirsurface molecule expression, such as CD45RA/RO/RB expression. Theresults show that small populations of cells, which may be involved in adisease condition such as cancer, can be detected and used for thediagnosis of MDS.

Example 5 A Small Population of Cells Responsive to Stem Cell Factor(SCF) Exist at Diagnosis and Expand During Disease Progression

The objective if this study is to identify cells in a diagnosis sampleand compare the results with a sample taken at a later time point fromthe same patient that will predict patient outcome. To achieve thisobjective myeloid populations were gated in the samples. Two dimensional(2D) plots are created for signaling analysis while three dimensionalplots (3D) are created for identifying cell lineage subsets. Gates aredrawn on cells with increase signaling to then back-gate to identifyphenotype of cells as determined by cell surface markers. This methodallows for the identification of differences in signaling betweendiagnosis and later time-point samples. The gates delineating cells withincreased signaling are applied to myeloid populations from independentstudies with AML samples.

Samples from AML patients were taken at diagnosis and at different timepoints after treatment. Cells in the samples were stimulated and stainedaccording to the detailed protocols described in Example 1. Differentpopulations of cells in the AML patients were compared at the time ofdiagnostics and at the time of relapse.

a. Gating of Flow Cytometry Data to Identify Live Cells and the Lymphoidand Myeloid Subpopulations:

Flow cytometry data can be analyzed using several commercially availablesoftware programs including FACSDiva™, FlowJo, and Winlist™. The initialgate is set on a two-parameter plot of forward light scatter (FSC)versus side light scatter (SSC) to gate on “all cells” and eliminatedebris and some dead cells from the analysis. A second gate is set onthe “live cells” using a two-parameter plot of Amine Aqua (a dye thatbrightly stains dead cells, commercially available from Invitrogen)versus SSC to exclude dead cells from the analysis. Subsequent gates arebe set using antibodies that recognize cell surface markers and in sodoing define cell sub-sets within the entire population. A third gate isset to separate lymphocytes from all myeloid cells (acute myeloidleukemia cells reside in the myeloid gate). This is done using atwo-parameter plot of CD45 (a cell surface antigen found on all whiteblood cells) versus SSC. The lymphocytes are identified by theircharacteristic high CD45 expression and low SSC. The myeloid populationtypically has lower CD45 expression and a higher SSC signal allowingthese different populations to be discriminated. The gated regioncontaining the entire myeloid population is also referred to as the P1gate.

b. Phenotypic Gating to Identify Subpopulations of Acute MyeloidLeukemia Cells:

The antibodies used to identify subpopulations of AML blast cells areCD34, CD33, and CD11b. The CD34⁺ CD11b⁻ blast population represents themost immature phenotype of AML blast cells. This population is gated onCD34 high and CD11b negative cells using a two-parameter plot of CD34versus CD11b. The CD33 and CD11b antigens are used to identify AML blastcells at different stages of monocytic differentiation. All cells thatfall outside of the CD34⁺CD11b⁻ gate described above (called “NotCD34+”) are used to generate a two-parameter plot of CD33 versus CD11b.The CD33⁺ CD 11b^(hi) myeloid population represents the mostdifferentiated monocytic phenotype. The CD33⁺CD11b^(intermediate) andCD33⁺CD11b^(lo) populations represent less differentiated monocyticphenotypes.

c. Back Gating to Identify the Phenotype of G-CSF and SCF ResponsiveCells:

A two-parameter or 3-parameter (3-D) plot was generated from the P1 gate(all myeloid cells). For G-CSF stimulation, the signaling responsesmeasured were p-Stat1, p-Stat3, and p-Stat5. The 3-D plot of p-Stat1 vs.p-Stat3 vs. pStat5 was generated in Spotfire. The two-parameter plotswere generated in FlowJo.

The data files for the unstimulated control sample and the G-CSF treatedsample were overlaid for comparison. In the results discussed below, thepaired patient samples at diagnosis (MDL-7) and at relapse (MDL-8) areshown. On the 3-D plot, the G-CSF responsive population was readilyvisible as a p-Stat5 positive population (See FIG. 17). A gate was seton the p-Stat5 positive population and was used to back gate onto a 3-Dplot of CD34 vs. CD33 vs. CD11b generated from the P1 gate. The datashows that the G-CSF responsive cells were found mainly in the CD33⁺CD11b⁻ population and that in the relapse sample there was an increasein G-CSF responsive cells within the CD33⁺CD11b⁻ population (4% atdiagnosis compared to 27% at relapse). Analysis of G-CSF responsivepopulations in healthy bone marrow showed that the responding cells aremainly CD34⁺.

d. Results

In this CR relapse patient two samples are available for analysis. Onesample was taken at the time of diagnosis and the second was taken about4 months later when the patient relapsed. The samples were measured fortheir basal phosphorylated Stat-5 (p-Stat5) and Stat-1 (p-Stat1) and thephosphorylated levels in response to IL-27 and G-CSF (FIG. 16). FIG. 16shows an example of a bone marrow sample at diagnosis and relapse from a34 year old patient whose response was CR Relapse with M2 AML and Flt3ITD+. Comparison of the two samples revealed more p-Stat5 and p-Stat1 inthe samples taken at relapse. FIG. 16 shows that at diagnostics there isa small sample that show levels of p-Stat-5 in response to G-CSF. Thispopulation is increased at relapse (See arrow in FIG. 16).

In addition, the samples were evaluated for their basal levels ofphosphorylated Akt (p-Akt) and ribosomal S6 protein (p-S6) (FIG. 17).FIG. 17 shows an example of results in a bone marrow sample at diagnosisand post induction treatment from a 68 year old patient who was arefractory to induction therapy and therefore classified as anon-responder (NR) and with M5 AML and Flt3R wild-type. Comparison ofthe two samples revealed more p-Akt and p-S6 in the samples taken atrelapse. The two samples were also treated with stem cell factor (SCF)and FLT3L and the signaling response was evaluated by determining thelevels of p-Akt and p-S6. In the sample taken at diagnosis, a smallpopulation of cells showed a response to SCF and the dots in the gateshow cells with an increase in p-Akt and p-S6 (See FIG. 17). However,there was a far greater increase in the SCF-mediated increase in p-Aktand p-S6 in the sample taken at relapse. Back-gating revealed thephenotype of the responding cell population which was identified as amyeloid cell sub-set defined by CD33+, CD11b−, CD34−. Table 3 describesthe phenotypes of the SCF-responsive cells

TABLE 3 Phenotype of SCF Subject Responsive Cell Subsets AML Patient 1CD34+, CD33−, CD11b− AML Patient 2 CD34+, CD33+, CD11b− AML Patient 3CD34−, CD33+, CD11b− Healthy CD34+, CD33−, CD11b−

These responding cells did not respond as robustly to FLT3 ligandstimulation. However, it is clear that there is a small population ofSCF responsive (double positive) cells in the sample at diagnosis. Thisfinding was seen in all the patients with matched (DX and Relapse)samples (n=3).

In order to predict whether the presence of a small population of SCFresponsive (p-Akt/p-S6) double positive population at diagnosis couldpredict outcome, a gate that delineated the double positive populationwas applied to a set of historical phosphoflow data from a set of AMLsamples taken at diagnosis and evaluated for SCF signaling in anindependent study (FIG. 18). FIG. 18 shows results from the bone marrowof a CR relapse 34 year old patient with M2 AML and Flt3 ITD+. FIG. 19depicts the results for the SCF responsive (p-Akt/p-S6) double positivepopulation in the set of AML patients. The results show that 9/10patients with an SCF responding double positive cell frequency of >3%relapsed within two years (FIG. 19). Only one patient in which there wasan SCF-responding double population had a complete clinical response(CCR). Furthermore, only a small number of cells were necessary tostratify these patients. As shown in slide 5, in one particular patient,183 double positive cells were captured.

To summarize, in this small patient subset 3/3 evaluated patients hadthe double positive SCF responding cells. As mentioned above, in anindependent study with a larger number of AML patient samples taken atdiagnosis, 9/10 patients with an SCF responding double positive cellfrequency of >3% relapsed within two years (FIG. 19). Notably, not allof the patients that had a poor outcome exhibited this SCF response. Thecell surface phenotype of the double positives are generally negativefor CD11b surface protein, but can be either CD34 positive, CD33positive, or a combination of both (see Table 3). This contrasts withhealthy bone marrow in which the SCF responsive cells are restricted tothe CD34+ subset.

When the analysis using the same gate was performed in peripheral bloodmononuclear cells (PBMCs) from AML patients, a trend similar to the bonemarrow data was seen (data not shown). Since SCF-responsive cells arenot present in the blood circulation of healthy subjects, PBMCs or wholeperipheral blood may be a preferred source of cells for an assay thatmeasures the SCF responsive double positives since background “assaynoise” could be avoided. It would be predicted that any SCF signalingwould emanate from the diseased cells.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method of predicting a change in a health status in an individualfrom a first state to a second state comprising: (a) determining thepresence of a first and second class of cells in a sample from saidindividual said presence being determined by a method comprisingdetermining an activation level of an intracellular activatable elementin single cells from said sample; (b) classifying said single cells intosaid first and second class, wherein at least one class is classifiedbased on said activation level; (c) calculating a ratio of said firstand second class of cells; and (d) predicting a change in a healthstatus in said individual from a first state to a second state when saidratio exceeds a threshold number.
 2. The method of claim 1, wherein saidclasses are predefined classes.
 3. The method of claim 1, wherein saidthreshold number is a predetermined threshold number, wherein saidpredetermined threshold number has been associated with said secondstate.
 4. The method of claim 1, wherein said second state is thelocation of an individual on a continuum that comprises normal,pre-pathological, and pathological states.
 5. The method of claim 4,wherein said continuum is a continuum wherein the pathological state isan immunologic, malignant, or proliferative disorder or a combinationthereof.
 6. The method of claim 5, wherein the malignant disorder is asolid tumor or a hematologic malignancy.
 7. The method of claim 5,wherein said malignant disorder is non-B cell lineage derived.
 8. Themethod of claim 7, wherein said non-B cell lineage derived malignantdisorder is selected from the group consisting of Acute myeloid leukemia(AML), Chronic Myeloid Leukemia (CML), non-B cell Acute lymphocyticleukemia (ALL), non-B cell lymphomas, myelodysplastic disorders,myeloproliferative disorders, myelofibroses, polycythemias,thrombocythemias, and non-B cell atypical immune lymphoproliferations.9. The method of claim 5, wherein said malignant disorder is a B cell orB cell lineage derived disorder.
 10. The method of claim 9, wherein saidmalignant disorder is a B-Cell or B cell lineage derived disorderselected from the group consisting of Chronic Lymphocytic Leukemia(CLL), B cell lymphocyte lineage leukemia, B cell lymphocyte lineagelymphoma, Multiple Myeloma, and plasma cell disorders
 11. The method ofclaim 1, further comprising predicting a response to a treatment for apre-pathological or pathological condition, or a response to treatmentfor a pre-pathological or pathological condition.
 12. The method ofclaim 1, wherein the activation levels of a plurality of intracellularactivatable elements in single cells is determined.
 13. The method ofclaim 1, wherein said plurality of cells obtained from said individualis first exposed to a modulator before determining said activation levelof said activatable element.
 14. The method of claim 13, wherein saidmodulator is an activator or an inhibitor.
 15. The method of claim 14,wherein said modulator is a growth factor, cytokine, adhesion moleculemodulator, hormone, small molecule, polynucleotide, antibody, naturalcompound, lactone, chemotherapeutic agent, immune modulator,carbohydrate, protease, ion, reactive oxygen species, or radiation. 16.The method of claim 1 wherein the sample is a blood sample, a biopsysample or a surgical sample.
 17. The method of claim 1, wherein theclass is a class of cells wherein one or more activation levels of thecells are different when compared to normal control values, or whencompared to previous determinations made in a series of samples fromsaid individual.
 18. The method of claim 1, wherein said predicting achange in said health status in said individual is performed on aplurality of samples from said individual.
 19. The method of claim 18,wherein said plurality of samples comprises samples from differentlocations in the individual, samples taken at different times from theindividual, samples treated in different ways prior to determining theactivation level, or a combination thereof.
 20. The method of claim 19,wherein the method further comprises determining the rate of change ofsaid ratio.
 21. The method of claim 20, wherein, said rate of change isexpressed as the doubling time of said cells.
 22. The method of claim 1,further comprising determining an appropriate course of treatment forsaid individual based on said status of the individual.
 23. The methodof claim 22, wherein said appropriate course of treatment compriseswatchful waiting, supportive care, initiating a therapy, not initiatinga therapy, stopping, shortening, prolonging, or modifying an existingtherapy, adding an additional therapy to existing therapy, orcombinations of the foregoing.
 24. The method of claim 22, wherein saidtherapy is selected from the group consisting of surgical excision,transplantation, or the administration of a physical, chemical, orbiological agent, or combinations thereof.
 25. The method of claim 1,wherein one or more characteristics of the individual is determined, andthe change in health status in the individual is determined based onboth the ratio and the one or more characteristics of the individual.26. The method of claim 22 wherein said determining of an appropriatecourse of treatment is also based on one or more characteristics of theindividual.
 27. The method of claim 25, wherein said one or morecharacteristics is physical characteristics, clinical status, treatmentcharacteristics, biochemical/molecular markers or a combination thereof.28. The method of claim 1, wherein said activation level is based on theactivation state selected from the group consisting of extracellularprotease exposure, novel hetero-oligomer formation, glycosylation state,phosphorylation state, acetylation state, methylation state,biotinylation state, glutamylation state, glycylation state,hydroxylation state, isomerization state, prenylation state,myristoylation state, lipoylation state, phosphopantetheinylation state,sulfation state, ISGylation state, nitrosylation state, palmitoylationstate, SUMOylation state, ubiquitination state, neddylation state,citrullination state, deamidation state, disulfide bond formation state,proteolytic cleavage state, translocation state, changes in proteinturnover, multi-protein complex state, oxidation state, multi-lipidcomplex, and biochemical changes in cell membrane.
 29. The method ofclaim 28, wherein said activation state is a phosphorylation state. 30.The method of claim 1, wherein said classifying of said single cellsfurther comprises determining cell size, cell granularity, the presenceor absence of one or more cell surface markers, the presence or absenceof one or more intracellular markers, or combination thereof.
 31. Themethod of claim 30, wherein said cell surface markers and saidintracellular markers are independently selected from the groupconsisting of proteins, carbohydrates, lipids, nucleic acids andmetabolites.
 32. The method of claim 30, wherein said determining of thepresence or absence of one or more cell surface markers or intracellularmarkers comprises determining the presence or absence of an epitope inboth activated and non-activated forms of said one or more cell surfacemarkers or intracellular markers.
 33. The method of claim 30, whereinsaid activatable element is selected from the group consisting ofproteins, carbohydrates, lipids, nucleic acids and metabolites.
 34. Themethod of claim 33, wherein said activatable element is a protein. 35.The method of claim 34, wherein said protein is a protein subject tophosphorylation and/or dephosphorylation.
 36. The method of claim 34,wherein said protein is selected from the group consisting of kinases,phosphatases, lipid signaling molecules, adaptor/scaffold proteins,cytokines, cytokine regulators, ubiquitination enzymes, adhesionmolecules, cytoskeletal/contractile proteins, heterotrimeric G proteins,small molecular weight GTPases, guanine nucleotide exchange factors,GTPase activating proteins, caspases, proteins involved in apoptosis,cell cycle regulators, molecular chaperones, metabolic enzymes,vesicular transport proteins, hydroxylases, isomerases, deacetylases,methylases, demethylases, tumor suppressor genes, proteases, ionchannels, molecular transporters, transcription factors/DNA bindingfactors, regulators of transcription, and regulators of translation. 37.The method of claim 34, wherein said protein is selected from the groupconsisting of HER receptors, PDGF receptors, Kit receptor, FGFreceptors, Eph receptors, Trk receptors, IGF receptors, Insulinreceptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1,Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70,Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TGFβreceptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2,MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1, Wee1, Casein kinases, PDK1,SGK1, SGK2, SGK3, Akt1, Akt2, Akt3, p90Rsks, p70S6Kinase, Prks, PKCs,PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chk1,Chk2, LKB-1, MAPKAPKs, Pim1, Pim2, Pim3, IKKs, Cdks, Jnks, Erks, IKKs,GSK3a, GSK3p, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class 3,mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, Receptor proteintyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptortyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs),Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecularweight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases,Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C,PP1, PP5, inositol phosphatases, PTEN, SHIPs, myotubularins,phosphoinositide kinases, phopsholipases, prostaglandin synthases,5-lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffoldproteins, Shc, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP),SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nck, Grb2 associated binder(GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-Cellleukemia family, IL-2, IL-4, IL-8, IL-6, interferon γ, interferon α,suppressors of cytokine signaling (SOCs), Cbl, SCF ubiquitination ligasecomplex, APC/C, adhesion molecules, integrins, Immunoglobulin-likeadhesion molecules, selectins, cadherins, catenins, focal adhesionkinase, p130CAS, fodrin, actin, paxillin, myosin, myosin bindingproteins, tubulin, eg5/KSP, CENPs, β-adrenergic receptors, muscarinicreceptors, adenylyl cyclase receptors, small molecular weight GTPases,H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam,Sos, Dbl, PRK, TSC1,2, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2,Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Bcl-2, Mcl-1,Bcl-XL, Bcl-w, Bcl-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk,Noxa, Puma, IAPs, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D,Cyclin E, Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP, p21CIP, molecularchaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAaCarboxylase, ATP citrate lyase, nitric oxide synthase, caveolins,endosomal sorting complex required for transport (ESCRT) proteins,vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylasesPHD-1, 2 and 3, asparagine hydroxylase FIH transferases, Pin1 prolylisomerase, topoisomerases, deacetylases, Histone deacetylases, sirtuins,histone acetylases, CBP/P300 family, MYST family, ATF2, DNA methyltransferases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-1,p53, Hdm, PTEN, ubiquitin proteases, urokinase-type plasminogenactivator (uPA) and uPA receptor (uPAR) system, cathepsins,metalloproteinases, esterases, hydrolases, separase, potassium channels,sodium channels, multi-drug resistance proteins, P-Gycoprotein,nucleoside transporters, Ets, Elk, SMADs, Rel-A (p65-NFKB), CREB, NFAT,ATF-2, AFT, Myc, Fos, Sp1, Egr-1, T-bet, β-catenin, HIFs, FOXOs, E2Fs,SRFs, TCFs, Egr-1,1-catenin, FOXO STAT1, STAT 3, STAT 4, STAT 5, STAT 6,p53, WT-1, HMGA, pS6, 4EPB-1, eIF4E-binding protein, RNA polymerase,initiation factors, elongation factors.
 38. The method of claim 1,wherein said activation level is determined by a process comprising thebinding of a binding element which is specific to a particularactivation state of the particular activatable element.
 39. The methodof claim 38, wherein said binding element comprises an antibody.
 40. Themethod of claim 33, wherein said activatable element is responsive to achange in metabolic state, temperature, local ion concentration, orexpression of a heterologous protein.
 41. The method of claim 1, whereinthe step of finding the activation level comprises the use of flowcytometry, immunofluorescence, confocal microscopy,immunohistochemistry, immunoelectronmicroscopy, nucleic acidamplification, gene array, protein array, mass spectrometry, patchclamp, 2-dimensional gel electrophoresis, differential display gelelectrophoresis, microsphere-based multiplex protein assays, ELISA, andlabel-free cellular assays to determine the activation level of one ormore intracellular activatable element in single cells.
 42. The methodof claim 1 wherein said threshold number expressed as a percentage isabout 30%.
 43. The method of claim 1 wherein said threshold numberexpressed as a percentage is about 5%.
 44. The method of claim 1 whereinsaid threshold number expressed as cell frequency is about 10⁻⁴.